- Email: [email protected]
Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect Salmonella in pork and pork products
Journal Pre-proof Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect Salmonella in pork and pork products Kasanchi M. Momin, Arockiasamy Arun Prince Milton, Sandeep Ghatak, Shiny C. Thomas, Govindarajan Bhuvana Priya, Samir Das, Ingudam Shakuntala, Rajkumari Sanjukta, Kekungu-u Puro, Arnab Sen PII:
S0890-8508(19)30401-3
DOI:
https://doi.org/10.1016/j.mcp.2020.101510
Reference:
YMCPR 101510
To appear in:
Molecular and Cellular Probes
Received Date: 15 October 2019 Revised Date:
30 December 2019
Accepted Date: 13 January 2020
Please cite this article as: Momin KM, Prince Milton AA, Ghatak S, Thomas SC, Priya GB, Das S, Shakuntala I, Sanjukta R, Puro K-u, Sen A, Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect Salmonella in pork and pork products, Molecular and Cellular Probes (2020), doi: https://doi.org/10.1016/j.mcp.2020.101510. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Contribution of each author K.M. Momin, Project staff, DST PSR
A.A.P. Milton
S. Ghatak
S.C. Thomas
G.B. Priya, SRF, DBT AMR S. Das I. Shakuntala R.K. Sanjukta K. Puro A. Sen
Performed all the major experiments (Analytical sensitivity, LOD, Field applicability, Comparison assays) Conceptualized the work, designed the experiment plan, monitored all the experiments, analyzed results, drafted the manuscript Performed specificity experiment, provided critical inputs then and there required, drafted the manuscript Designed the PSR primers used in the study. Helped in incorporation of olignonucleotide sequences of exogenous (botanic) origin with PSR primers. Performed artificial spiking experiment in pork Analyzed the results and shared reference TB cultures. Helped in designing the experiment and gave critical inputs Helped in analyzing results and setting all the figures Helped in drafting the manuscript and gave critical inputs Helped in drafting the manuscript and proof reading of the manuscript
1 2 3 4 5 6 7 8 9 10 11
Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect Salmonella in pork and pork products Kasanchi M. Momin1,§, Arockiasamy Arun Prince Milton1,§,*, Sandeep Ghatak1,*, Shiny C. Thomas2, Govindarajan Bhuvana Priya1, Samir Das1, Ingudam Shakuntala1, Rajkumari Sanjukta1, Kekungu-u Puro1, Arnab Sen1 1
Division of Animal Health, Indian Council of Agricultural Research (ICAR) Research Complex for NEH Region, Umiam, Meghalaya 793103, India 2 School of Life Sciences, Assam Don Bosco University, Guwahati, Assam, India
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§: Both authors contributed equally to this work *Corresponding Authors: Dr. A.A.P Milton, Dr. S. Ghatak, Division of Animal Health,
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ICAR Research Complex for NEH Region, Umiam- 793 103, Meghalya.
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Email: [email protected]; [email protected]
16
Mobile: +91 8650918630; +919436380401
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Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect
33
Salmonella in pork and pork products
34
Abstract
35
The polymerase spiral reaction (PSR), a novel isothermal method for targeted DNA
36
amplification, was effectively applied to detect Salmonella in artificially spiked pork. The
37
specificity of the developed PSR was tested using 16 Salmonella and 15 non-Salmonella
38
strains. The PSR assay was 10-fold more sensitive than conventional end-point PCR, having
39
a sensitivity comparable to real-time PCR. The limit of detection of the developed assay was
40
4 X 103 per gram of pork without enrichment and 4 CFU per gram after a 6 h enrichment. The
41
detection of 4 CFU per gram of pork was achieved within 8 h. The PSR assay was successful,
42
and accurate in comparison to microbiological methods, in detecting Salmonella in 11 of 76
43
commercial pork samples. Therefore the positive predictive value, negative predictive value
44
and accuracy rate of the developed assay were 100%. Considering its rapidity, user-
45
friendliness, simplicity, cost-effectiveness and equipment-free nature, this PSR assay is a
46
promising tool for the food industry for the detection of Salmonella and prevention of
47
Salmonella outbreaks and recalls.
48
Keywords: Salmonella; Polymerase spiral reaction; Pork; Pathogen detection
49
1. Introduction
50
Salmonella is an important foodborne pathogen and the primary cause of bacterial
51
foodborne illnesses worldwide [1]. It causes bacterial gastroenteritis and is responsible for
52
approximately 30% of food poisoning cases in the USA (1.4 million human cases annually)
53
[2]. Globally, non-typhoidal Salmonella account for approximately 153 million cases
54
(approx) of gastroenteritis and 57,000 deaths each year [3]. The most common sources of
55
Salmonella infection are foods of animal origin. Contaminated food products such as eggs,
56
poultry, pork and beef contribute to 75 % of human Salmonella cases [4]. While data on the
57
prevalence of Salmonella spp. in foods of animal origin in India is patchy, several reports
58
indicate the public health importance of the organism [3, 5-7].
59
In healthy humans, the infectious dose is typically ≥105 Salmonella cells, but in
60
extremely susceptible individuals, as little as 15-20 cells can cause disease [8]. When present,
61
foodborne pathogens are often at very low numbers relative to background microflora,
62
making their detection and identification difficult [9]. Conventional detection methods for
63
Salmonella spp. include pre-enrichment, selective enrichment and plating on selective media
64
followed by biochemical and serological testing of presumptive isolates. While these methods
65
are highly specific and are considered as the gold standard, they are time-consuming
66
(requiring 7-10 days) and technically demanding (requiring well-trained manpower) [10]. In
67
spite of the many advantages of nucleic acid based detection methods such as PCR, and real-
68
time PCR, these methods impose many other constraints that prevent their routine use in
69
resource-limited settings (e.g., costly equipment and laboratory set-up, post-PCR processing,
70
technical training, etc.) [11]. Most of these detection methods are not appropriate for onsite
71
detection or small scale food establishments. Therefore, rapid, cost-effective, simple,
72
sensitive, specific and field portable assays are crucial for the detection of foodborne
73
pathogens to control the distribution of contaminated foods [12].
74
In recent years, many isothermal nucleic acid amplification-based assays, have been
75
developed and deployed to detect foodborne pathogens. These methods can synthesize a large
76
amount of DNA rapidly with high specificity. These assays obviate the necessity for a
77
thermocycler, gel electrophoresis unit and trained personnel. Among them, loop-mediated
78
isothermal amplification (LAMP) has been broadly applied and has been demonstrated to be
79
a powerful tool for the detection of Salmonella in foods [11-19]. Invasin A gene (invA) is the
80
most common target for nucleic acid based detection of Salmonella. The invA gene is
81
chromosomally located and encodes for an inner membrane protein of Salmonella that is
82
essential for the invasion of epithelial cells and is conserved in all the Salmonella serotypes
83
[20]. Despite the many advantages of LAMP, the technique has some important limitations
84
(the requirement for multiple primers, requiring rigorous optimization and the possibility of
85
carryover or leftover contamination leading to false-positive results) that have limited its
86
adoption [21].
87
Polymerase Spiral Reaction (PSR) is a novel isothermal assay which rapidly amplifies
88
target nucleic acid in the temperature range of 60–650C with high sensitivity and specificity.
89
In contrast to LAMP, it requires only two primers [22]. The technique does not require any
90
sophisticated instruments and has successfully been demonstrated as a potential point-of-care
91
test for detecting many pathogens of medical and veterinary significance [23-28]. However, it
92
has seldom been applied for the detection of foodborne pathogens and has yet to be adopted
93
in the food industry. More recently, a PSR assay for detection of Salmonella in food was
94
standardized [29]. However the main limitations of the study are that they have used two pair
95
of primers, artificial spiking experiment was not performed to determine the limit of detection
96
(LOD) and the effect of enrichment in improving the LOD was not studied.
97
Therefore, the present study aimed to develop and evaluate a PSR assay targeting the
98
invA gene to detect Salmonella in foods. The developed assay was validated by detecting
99
Salmonella in artificially contaminated pork. The effect of a short culture enrichment on
100
detection limit of Salmonella by PSR was also studied. The sensitivity and specificity of the
101
developed PSR were also compared to PCR and real-time PCR methods. To avoid primer
102
bias, PSR assay was compared with newly optimised PCR and real-time PCR assays
103
targeting same conserved region of invA gene of Salmonella. We chose to use same primers
104
(except the botanical portion of the PSR primers) in all three assays (PCR, real-time PCR and
105
PSR) as our aim was to compare assays, not primers. However analytical sensitivity of PCR
106
and real-time PCR assays based on well-cited primers were also compared with the current
107
PSR assay. The portability of the developed assay was also demonstrated by screening field
108
pork samples.
109
2. Materials and Methods
110
2.1 Bacterial strains
111
Five Salmonella reference strains, 11 Salmonella strains isolated from food samples
112
and 15 non-Salmonella strains (Table 1) were used in the present study. The strains were
113
obtained from American Type Culture Collection (Manassas, Virginia, USA) and the
114
National Salmonella Centre (Indian Veterinary Research Institute, Bareilly, Uttar Pradesh,
115
India). Bacterial strains were stored as 20% glycerol stocks at -80°C. Strains were streaked
116
on suitable media (HiMedia, India) and grown at 37 °C. Broth cultures were inoculated from
117
single colonies into 10 mL of appropriate media (HiMedia, India) and grown overnight at 37
118
°C.
119
Typhimurium ATCC 51812 was used as a reference strain in the analytical sensitivity and
120
meat spiking studies.
121
Table 1. Bacterial strains used in the study
Cells were harvested and used to prepare DNA as described below. Salmonella
Bacterial Species
Salmonella Strains Salmonella Typhimurium Salmonella Enteritidis Salmonella Paratyphi Salmonella Pullonum Salmonella Uccle Salmonella food isolates (n=11) non-Salmonella Strains Shigella boydii Shigella sonnei Escherichia coli Enterococcus faecalis Pseudomonas aeruginosa
Strain/ Source*
ATCC 51812 NSC 2478 NSC 77 NSC E79 NSC 60a
ATCC 25931 ATCC 9207 ATCC 25922 ATCC 51299 ATCC 10145
122
Listeria monocytogenes ATCC 13119 Klebsiella pneumonia ATCC 700608 Klebsiellaoxytoca ATCC 43863 Campylobacter jejuni ATCC 33291 Staphylococcus aureus ATCC 33591 Staphylococcus xylosus ATCC 29971 Staphylococcus epidermidis ATCC 12228 Clostridium perfringens ATCC 13124 Mycobacterium smegmatis ATCC 607 Mycobacterium bovis AN5 *ATCC-American Type Culture Collection (USA); NSC-National Salmonella Centre (India)
123
2.2 Extraction of genomic DNA
124
Genomic DNA of both Salmonella and non-Salmonella strains was isolated using
125
QIAmp DNA Mini Kit (Qiagen, Germany) as per manufacturer’s instructions. The
126
concentration of the extracted DNA were measured using UV-Visible spectrophotometry
127
(Nanodrop,Thermo Scientific, USA). The DNA preparations were stored at -20 °C.
128
2.3 Designing of PSR primers
129
The PSR primers were designed to target the conserved region of the invA gene (Gen
130
Bank Accession number CP043222) of Salmonella using the Primer3 program (NCBI). The
131
principles of PSR primer design were described previously [22]. The Primer3 suggested
132
primers
133
(https://blast.ncbi.nlm.nih.gov/Blast.cgi). The selected primers were synthesized by Imperial
134
Life Scienes (Haryana, India). The in-house PCR and real-time PCR targeting the invA gene
135
were used to compare the sensitivity and specificity of developed PSR assay (data not
136
shown). The primer sequences designed and used for PSR, PCR and realtime PCR are given
137
in Table 2.
138
Table 2. Primer sequences used in the study
Assay
were
checked
for
specificity
in
silico
with
the
BLAST-N
Primer
Sequences
INV2PF
5’-acgattcgtacatagaagtatagTGGATTTGTCCTCCGCCCTG-3’
program
Product size Variable
Source This study
PSR PCR & realtime PCR
INV2PR
5’-gatatgaagatacatgcttagcaCCGTATCGCCATTTACGCGG-3’
INV2F
5’-TGGATTTGTCCTCCGCCCTG-3’
INV2R
5’-CCGTATCGCCATTTACGCGG-3’
This study This study 129 bp This study
139 140
2.4 Optimization of the PSR and visual detection of amplified products
141
The PSR assay was optimized for assay temperature (60-70 °C) and time (15-90 min)
142
as well as dNTP (0.5-1.6 mM) (Thermoscientific, USA), MgSO4 (2.0-8.0 mM) (New England
143
BioLabs, USA), betaine (0.5-1.4mM) (Sigma-Aldrich, USA), BST 2.0 Warm start
144
polymerase (4-12 U) (New England BioLabs, USA) and primer (5µM-15µM)concentrations.
145
PCR and real-time PCR assays were optimized to compare the PSR assay. The species-
146
specific primer sets for Salmonella enterica were designed from the same conserved region
147
of invA gene chosen for PSR (Table 2). The PCR reaction mixture (total volume, 25 µl)
148
contained 12.5 µl of 2x Dream Taq Master Mix (Dream Taq DNA polymerase, 2X Dream
149
Taq Green buffer, 0.4mM dNTPs, 4mM MgCl2), 1µl of 10pmol each of forward and reverse
150
primer, 1 µl of the template DNA and 8.5µl nuclease-free water. Amplification was
151
performed in Mastercycler nexus GX2 (Eppendorf, Germany). The cycling conditions
152
consisted of 10 min initial denaturation at 94 °C, followed by 35 cycles each of 1min
153
denaturation at 94 °C, 1.5 min annealing at 62 °C and 1min elongation at 72 °C and a final
154
extension step of 5 min at 72 °C. Real-time PCR was carried out in a 20µl reaction volume
155
consisting of 10µl Power SYBR Green PCR Master Mix (Applied Biosystems), 0.1µl each of
156
forward and reverse primer, 7.8µl of nuclease-free water and 1µl sample DNA. The reaction
157
was carried out in 7500 Fast Real-Time PCR System (Applied Biosystems) in following
158
conditions: initial denaturation at 95 °C for 10 minutes, followed by 40 cycles each of 15 sec
159
denaturation at 94 °C, 1 min annealing at 60 °C and 1min elongation at 72 °C and a final
160
extension step of 5 min at 72 °C. The real-time PCR conditions consisted of an initial step of
161
95°C for 10 minutes followed by an amplification program for 40 cycles of 15 seconds at
162
95°C, 1 minute at 60°C, 1 minute at 72°C with fluorescence acquisition at the end of each
163
extension. The amplification program was immediately followed by a melt program
164
consisting of 15 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 95°C with
165
fluorescence acquisition at each temperature transition. The PCR amplicon was detected by
166
electrophoresis on 1.5% agarose gel stained with ethidium bromide (0.5 µg/ml) and
167
photographed using gel imaging system (AlphaImager, UK). The amplified PSR products
168
were detected visually (under white light) by the addition of 1 µl of SYBR Green I dye
169
(10,000 X, Sigma-Aldrich, USA) diluted 1:10 in 1X phosphate buffered saline (PBS) which
170
produced fluorescent green colour in positive whereas the colour remained unchanged
171
(orange colour) in the negative reactions. The PSR products were also subjected to agarose
172
gel electrophoresis similar to PCR products and photographed for confirmation.
173 174
2.5 Analytical sensitivity and specificity
175
To determine the analytical sensitivity, serial tenfold dilutions (10-1 to 10-8) of the
176
extracted genomic DNA of Salmonella Typhimurium ATCC 51812 were prepared and used
177
to perform PSR, PCR and real-time PCR as per the standardized protocols. In real-time PCR,
178
a Ct value of 32 was set as a cut-off point. Analytical sensitivity of the PSR assay was also
179
compared with the previously published invA based PCR [30] and real-time PCR assays [31].
180
The specificity of the developed PSR assay was determined by checking for its cross-
181
reactivity with genomic DNA of 16 Salmonella and 15 non-Salmonella species/strains of 10
182
different genera.
183
2.6 Artificial contamination study and limit of detection (LOD) of the PSR assay
184
The limit of detection (LOD) of the PSR assay was estimated using artificially
185
contaminated pork as a model meat. A single S. Typhimurium (ATCC 51812) colony was
186
picked from an XLD agar (HiMedia, India) plate and was cultured in 10 ml of tryptone soy
187
broth (HiMedia, India) at 37 °C overnight (16 hr). After the overnight incubation, the
188
Salmonella cells were harvested by centrifugation at 10,000 x g for 10 min. The bacterial
189
pellet was washed once with 2 ml of PBS and re-suspended in final volume of 2 ml PBS.
190
Serial 10 fold dilutions (10-1 to 10-8) of this bacterial suspension were made in 1X sterile
191
PBS. The bacterial concentration in overnight culture was deteremined by spreading 100 µl
192
of serially diluted suspensions onto XLD agar plates in duplicate followed by overnight (18
193
h) incubation at 37 °C. The pork cut was purchased from the local market in Umiam,
194
Meghalaya. The pork was confirmed to be free from Salmonella by real-time PCR and a
195
microbiological culture method (ISO 6579:2002; [32]). Pork homogenate was prepared by
196
stomaching 25 g of meat in 225 ml of 0.1% buffered peptone water (HiMedia, India) and 9
197
ml of the homogenates were distributed in 15 ml culture tubes. One ml of each dilution of the
198
prepared bacterial suspension was inoculated into 9 ml of the pork homogenate dispensed in
199
15 ml culture tubes. A negative control was created by mixing 9 ml of the meat homogenate
200
with 1 ml sterile 1X PBS. Genomic DNA was extracted using the DNeasy blood and tissue
201
kit (Qiagen, Germany) following the manufacturer’s protocol. Conventional PCR, real-time
202
PCR and PSR assays were performed using extracted DNA samples.
203
2.7 Evaluation of the effect of enrichment on LOD of the PSR assay
204
To evaluate the effect of enrichment, samples from each dilution of inoculated pork
205
were collected after 6h of incubation at 37 °C. DNA was extracted from enriched samples.
206
Briefly, 2 ml of the sample was collected from each dilution of spiked pork homogenate at 0
207
h (without enrichment) and after 6 h of enrichment and centrifuged at 10,000 g for 5 min. The
208
resulting cell pellet was then re-suspended in 180 µl of lysis buffer and was processed for
209
genomic DNA extraction as per manufacturer’s protocol (Qiagen, Germany). Extracted DNA
210
was used as the template in PCR, real-time PCR and PSR assays and the LOD were
211
compared between the assays. All experiments using artificially contaminated pork were
212
done in triplicate.
213
2.8. Application of the developed PSR assay to field samples
214
To evaluate applicability of the developed PSR assay to naturally contaminated samples,
215
it was deployed to screen raw pork and processed pork samples (n=76) for the presence of
216
Salmonella from the retail market of Meghalaya. Samples were obtained from the local wet
217
markets of Meghalaya. The accuracy of the developed PSR assay was compared with the
218
cultural methods, conventional PCR and real-time PCR. Salmonella isolation and
219
identification was done according to ISO 6579:2002 methods [32]. Additional confirmation
220
of all isolates was done by PCR targeting the invA gene.
221
2.9 Statistical analysis
222
The performances of the newly developed diagnostic PSR, PCR and real-time PCR
223
assays were evaluated by determining the sensitivity, specificity, Positive Predictive Value
224
(PPV), Negative Predictive Value (NPV) and Accuracy. Calculations were based on the final
225
detection for each Salmonella isolate tested. True positive and true negative samples were
226
based on the results of Salmonella isolation and identification employing the ISO 6579:2002
227
method. PPV, NPV and accuracy were calculated by the following formulae
228 229 230 231 232 233 234 235 236 237 238 239
PPV=
(Number of TP) (Number of TP + Number of FP)
x 100
NPV=
(Number of TN) (Number of TN + Number of FN)
x 100
Accuracy=
(Number of TP + TN) (Number of TP + FP + TN + FN)
x 100
Where - TP: true positive, FP: false positive, TN: true negative, FN: false negative
240
3. Results
241
3.1 Optimization of the PSR assay
242
PSR was successfully optimized for all the reagents, temperature and time (Figures
243
1A-G). The optimized PSR mixture contained 2.5 µl of 10X Isothermal amplification buffer
244
(New England BioLabs, USA), 8.0 mM MgSO4, 1.4 mM dNTP’s, 0.8 M betaine,8.0 U Bst
245
2.0 WarmStart DNA polymerase, 10 µM forward primer, 10 µM reverse primer,1.0 µl of
246
template genomic DNA and nuclease-free water to make up the volume to 25 µl. The reaction
247
was incubated at 64 °C for 60 min. PSR amplicons were detected by the addition of SYBR
248
Green I dye and UV illumination. Positive samples yielded green fluorescence, whereas
249
negative reactions remained the original orange colour. The PSR amplicons were also
250
subjected to agarose gel electrophoresis which revealed a ladder pattern when stain with
251
ethidium bromide (Figures 1A-G).
252
3.2 Specificity or exclusivity of the PSR assay
253
The developed PSR assay for Salmonella targeting the invA gene demonstrated a high
254
degree of specificity, amplifying the genomic DNA of all 16 Salmonella strains tested and
255
yielding negative results with genomic DNA the other 15 bacterial species tested. The results
256
of specificity are presented in figure (Figure 2).
257
3.3 Analytical sensitivity of the PSR, PCR and real-time PCR assays
258
The analytical sensitivity of the developed PSR assay was determined and compared
259
with the results of newly developed and published PCR and real-time PCR assays. The
260
genomic DNA extracted from the pure culture of S. Typhimurium (ATCC 51812) had a
261
concentration of 100 ng/µl. The DNA concentration of ten-fold serial dilutions ranged from
262
10 ng/µl (100X10-1 ng/µl) to 1fg/µl (100X10-8 ng/µl). Since 1 µl of template DNA solution
263
was used in each amplification reaction, the total DNA per assay ranged from 10 ng to 1 fg.
264
The analytical sensitivity of newly developed PSR, real-time PCR and PCR assays were
265
found to be 100 fg, 100 fg and 1 pg, respectively indicating PSR to be 10 times more
266
sensitive than PCR and comparable to real-time PCR (Fig 3). The analytical sensitivity of
267
current PSR, previously published real-time PCR and PCR assays were found to be 100 fg, 1
268
pg and 10 pg, respectively indicating PSR to be 100 times more sensitive than previously
269
published PCR and 10 times sensitive than real-time PCR (Fig 4).
270
3.4 Artificial contamination study and limit of detection (LOD) of PSR,PCR and real-time
271
PCR assays
272
The bacterial concentration in the initial culture of S. Typhimurium ATCC 51812 was
273
4 x 107 CFU/ml. The homogenized pork (9 ml) was inoculated with 1 ml of 10-fold serially
274
diluted S. Typhimurium (ATCC 51812) culture prepared in 1X PBS. Therefore, inoculated
275
pork was carrying S. Typhimurium ranging from 4 x 106 CFU to 0.04 CFU of Salmonella in
276
the absence of culture enrichment, the detection limit of PCR, real-time PCR and PSR was
277
found to be 4 x 104 CFU/g, 4 x 103 CFU/g and 4 x 103 CFU/g of meat, respectively (Fig 5).
278
After enrichment for 6 h, the detection limit of PCR, real-time PCR and PSR was found to be
279
4 x 102 CFU/g, 40 CFU/g and 4 CFU/g of meat, respectively (Fig 6). The negative control did
280
not show any amplification, further confirming the specificity of developed PSR assay.
281
3.5 Real-world applicability of developed PSR assay
282
To evaluate real-world applicability of the developed PSR assay, it was deployed to
283
screen Salmonella from the raw, processed and cooked pork samples (n=76) collected from
284
retail markets of Meghalaya. The samples were screened with conventional PCR, real-time
285
PCR, PSR and conventional cultural methods. Out of 76 samples, following ISO 6579:2002
286
cultural method, Salmonella could be isolated from 11 pork samples. Without enrichment (at
287
0 hours), none of the samples gave positive results with conventional PCR, real-time PCR
288
and PSR assays. With a brief enrichment of 6 hours, PCR, real-time PCR and PSR could
289
detect Salmonella from 8, 11 and 11 samples, respectively.
290
3.6 Estimation of sensitivity, specificity, PPV, NPV and accuracy
291 292 293 294 295
The sensitivity, specificity, PPV, NPV and accuracy of the PSR, PCR and real-time PCR are furnished in the table 3.
296
Table 3. PPV, NPV, and accuracy (expressed as percentage, %) of PSR, PCR and real-
297
time PCR assays compared to ISO 6579:2002 Assays
298
Sensitivity Specificity PPV NPV Accuracy (95% LCL- (95% LCL- (95% LCL-95% (95% LCL- (95% LCL95% UCL) 95% UCL) UCL) 95% UCL) 95% UCL) PSR 100 100 100 100 100 (67.8-100.0) (93.0-100.0) (67.8-100.0) (93.0-100.0) (95.2-100.0) PCR 72 100 100 95.6 96.0 (39.3-92.7) (93-100.0) (59.7-100.0) (86.8-98.8) (88.9-99.1) Real-time 100 100 100 100 100 PCR (67.8-100.0) (93.0-100.0) (67.8-100.0) (93.0-100.0) (95.2-100.0) LCL, lower confidence limit; UCL, upper confidence limit
299
The PPV and NPV of the newly developed PSR assay for detection of Salmonella was 100%.
300
The accuracy of the newly developed PSR assay was also calculated to be 100%.
301
4. Discussion
302
Foodborne non-typhoidal salmonellosis remains an important economic and public health
303
burden for both developing and developed countries [3]. Most of the existing diagnostic
304
methods to detect Salmonella in foods suffer from many shortcomings, for example, they are
305
time consuming and expensive, exhibit low sensitivity, require trained manpower, yield a
306
high rate of false positivities (due to dead bacteria) etc. Considering the speed at which the
307
food moves from farm to fork and how rapidly an outbreak of human salmonellosis can
308
occur, rapid diagnostic methods are the clear priority and an urgent need from the food safety
309
and public health perspective. The rapidity in detecting the causative agent is crucial not only
310
for preventing hospitalization or clinical complication, but also for spotting the source of the
311
outbreak. This helps in efficient removal of the contaminated food products from the food
312
distribution chain. Ideally, pathogen detection would occur prior to distribution and
313
consumption of contaminated foods, reducing the occurrence of a food borne outbreaks.
314
Moreover, time consuming techniques such as conventional cultural methods (ISO 6579:
315
2002) [32] are not suitable for foods with a short shelf life like meat or ready to eat foods
316
[33]. In developing countries, the food regulatory agencies usually do not have sophisticated
317
laboratories, therefore simple techniques without the need for costly equipment and trained
318
manpower is a vital requirement.
319
Considering all this, we have developed a simple, rapid and promising polymerase spiral
320
reaction (PSR) assay to detect Salmonella in foods of animal origin. Moreover, we
321
demonstrated the potential of the PSR assay in rapid and accurate detection (<8 hours) of
322
Salmonella from raw pork and pork products. LAMP assays are frequently touted as simple
323
and rapid nucleic acid-based method for the detection of pathogens [8]. Furthermore, several
324
LAMP-based assays have been developed for detecting Salmonella from different foods of
325
animal origin [12, 14, 15, 34, 35]. The novelty of the present PSR assay in comparison with
326
LAMP assays is the requirement of only a single pair of primers that simplifies assay
327
development and eliminates the demand of rigorous optimization. Compared to LAMP, the
328
need for only two primers in PSR minimizes the likelihood of non-specific amplification and
329
contamination [27]. Thus, PSR is a unique blend of an isothermal amplification technique
330
and conventional PCR (single pair of primer).
331
The present PSR assay to detect Salmonella proved to be superior to conventional PCR as
332
it does not require a thermocycler, reducing the need for post PCR processing and has higher
333
sensitivity. Bst polymerase used in PSR and LAMP is shown to be more resistant to the
334
inhibitors of Taq DNA polymerase used in conventional PCR [36]. In the present study, the
335
analytical sensitivity of PSR and conventional PCR were 100 femtograms and 1 picogram,
336
respectively. The limits of detection in artificially contaminated pork without culture
337
enrichment were 4 x 104 CFU/g and 4 x 103 CFU/g in PCR and PSR, respectively. This
338
indicates that the present PSR assay was 10 fold more sensitive than the PCR (without
339
enrichment). After enrichment for 6 h, the detection limits of PCR and PSR were found to be
340
4 x 103 CFU/g, and 40 CFU/g of meat, respectively. So after a brief enrichment of 6 hours,
341
the PSR performed 100 times better than the conventional PCR assay.
342
The developed PSR assay also has the advantage over real-time PCR in that PSR does not
343
required costly and bulky equipment for post-PCR analysis. Moreover, the present PSR
344
assay was found to have comparable analytical sensitivity with that of real-time PCR (100
345
femtograms of genomic DNA). Furthermore, in artificially contaminated pork without
346
enrichment, both the PSR and real-time PCR had the same detection limits (4 x 103 CFU/g of
347
meat) and, with a brief enrichment of 6 h, the PSR assay (40 CFU/g of meat) was 10 times
348
more sensitive than the real-time PCR assay (400 CFU/g of meat).
349
The analytical sensitivity of 100 femtograms in the present assay is in concordance with
350
the earlier EMA (ethidium monoazide) LAMP and standard LAMP assays developed by Lu
351
et al. [34] and Wang et al. [14], respectively for detection of Salmonella in raw chicken and
352
pork. The limit of detection of the present PSR assay was found to be better than the earlier
353
RT-LAMP assay developed by Techathuvanan et al [12] for the detection of Salmonella from
354
pork products. They have reported a detection limit of 102CFU/ 25 g after enrichment and 106
355
CFU/25 g without enrichment. The detection efficiency of our assay is also better than an
356
earlier RT-LAMP assay developed for pork processing environment samples, wherein a limit
357
of detection of 10 CFU/ml was reported [37]. The current assay was also found to be more
358
efficient and rapid than an earlier assay which reported a detection limit of 35 CFU of
359
Salmonella per 250 ml of minced pork and raw milk with the assay time of 24 h that
360
includedtime for sample enrichment [15]. Our assay is also more sensitive than a real-time
361
LAMP assay developed for detection of Salmonella which has detected 7 CFU/ml in the
362
artificially spiked chicken samples after enrichment of 6 h [35] that is only slightly less
363
sensitive than our detection limit of 4 CFU/g after a 6 h enrichment.
364
In recent years, few PSR assays have been developed to detect important pathogens.
365
Among them, most of the assays have revealed a 10 fold higher sensitivity than conventional
366
PCR assay [23-26], which is in line with our result. However, PSR assays to detect bovine
367
herpes virus [26], Brucella [27] and Mycoplasma synoviae [28] have shown 100 times greater
368
sensitivity than conventional PCR. Similar to our PSR assay, previously reported assays have
369
shown comparable sensitivity to real-time PCR [25, 27, 29].
370
In the evaluation of the developed PSR assay for its field applicability, naturally
371
contaminated or field pork samples were tested with conventional PCR, real-time PCR, PSR
372
and conventional cultural methods. The conventional cultural method revealed 11
373
Salmonella-positive samples from 76 samples tested. None of the other assays could detect
374
Salmonella when employed in unenriched samples. However, after enrichment for 6 h, PCR,
375
real-time PCR and PSR could detect Salmonella from 8, 11 and 11 samples, respectively.
376
Although the limit of detection of the PSR assay before enrichment in the artificial
377
contamination study was 4 x 103 CFU/g of pork, this negative result might be due to lower
378
levels of contamination (<4 x 103 CFU/g)in the field samples. As the initial count of
379
Salmonella in raw foods samples are usually low [38], it is suggested that the present PSR
380
assay be employed after a 6 h enrichment. This also ensures the detection of viable
381
Salmonella from the samples. Finally, the complete operation time of the developed PSR
382
assay including DNA extraction and the enrichment step can be completed in approximately
383
8 hour.
384
The main advantage of this study is that a novel, simple and rapid assay was developed to
385
detect Salmonella in pork and pork products in resource-limited settings. Our study evaluated
386
the effect of enrichment and compared the developed assay with conventional PCR and real-
387
time PCR assays. The field applicability was also evaluated by screening naturally
388
contaminated or field samples and comparison was done with the gold standard cultural
389
method(ISO 6579:2002) [32]. While the developed PSR assay gives accurate results in both
390
pork and pork products, additional studies will be necessary to extend its application to other
391
foods.
392
In conclusion, we successfully developed and evaluated novel PSR assay for detection
393
of Salmonella from foods, which complies with the “ASSURED (affordable, sensitive,
394
specific, user-friendly, robust and rapid, equipment-free, and deliverable)” concept proposed
395
by WHO for the development of diagnostic. Since our assay has multiple advantages over
396
many Salmonella diagnostic assays currently employed; we foresee that it has the potential to
397
become the assay of choice for routine detection of Salmonella in small or resource-limited
398
food testing laboratories.
399 400
Competing Interests
401
The authors declare that there are no competing interests
402
Acknowledgements
403
The authors are thankful to the Department of Science and Technology (DST),
404
Ministry of Science and Technology, New Delhi, Government of India, for sanctioning the
405
project (research grant no. SP/YO/570/2018(G)). The authors are also grateful to the
406
Director, ICAR Research Complex for NEH Region, Umiam, Meghalaya, India for providing
407
necessary facilities to conduct this research work. We sincerely acknowledge the contribution
408
of Dr George C Paoli, Research Microbiologist, USDA ARS in improving the manuscript.
409 410
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Figure legends
538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579
Fig. 1. (A-G). Optimization of reagents (A. Primer; B. dNTP; C. MgSo4; D. Betaine; E. Bst Polymerase; F. Temperature; G. Time) concentration for PSR assay. First row- Visual detection with SYBR Green I showing green fluorescence in amplified products and orange in tubes with no amplification. Second row- Electrophoretic pattern of PSR amplified products. (Lane M- 100 bp plus ladder; NTC- Non-template control) Fig. 2 Specificity of PSR assay. First row- Agarose gel electrophoresis of PSR products (Lane 1-15) showing no amplification in non- Salmonella DNA and amplification in Salmonella DNA (Lane 16-20). Second row- Visual detection of PSR products (corresponding tube numbers 1-15) showing no amplification in non- Salmonella DNA and showing amplification in Salmonella DNA (corresponding tube numbers 16-20) using SYBR Green dye. (Lane M- 100 bp plus ladder; NTC- Non-template control) Fig. 3. Analytical sensitivity A) Agarose gel electrophoresis of new conventional PCR showing analytical sensitivity, B) Agarose gel electrophoresis (2%) and visual detection of PSR products showing analytical sensitivity, (Lane M- 100bp plus ladder, NTC- Nontemplate control) C) Amplification curve showing analytical sensitivity of new real-time PCR Fig. 4. Analytical sensitivity A) Agarose gel electrophoresis of conventional PCR (previously published [30]) showing analytical sensitivity (Lane M- 100bp plus ladder, NTC- Nontemplate control), B) Amplification curve showing analytical sensitivity of real-time PCR (previously published [31]). Fig. 5. Limit of detection in artificially contaminated pork (at 0 hour). A) Agarose gel electrophoresis of conventional PCR showing limit of detection, B) Agarose gel electrophoresis (2%) of PSR products showing limit of detection (Lane 1-9: 4X106 CFU/g, 4X105 CFU/g, 4X104 CFU/g, 4X103 CFU/g, 4X102 CFU/g, 40CFU/g, 4CFU/g, 0.4CFU/g, 0.04 CFU/g, NTC – No template control) and in second row- Visual detection of PSR products by addition of SYBR Green I showing limit of detection (Tube 1-9: 4X106 CFU/g, 4X105 CFU/g, 4X104 CFU/g, 4X103 CFU/g, 4X102 CFU/g, 40CFU/g, 4CFU/g, 0.4CFU/g, 0.04 CFU/g, Tube 10- NTC); C) Amplification curve of different dilutions of contaminated meat showing amplification till 4X103CFU/g. Fig. 6. Limit of detection in artificially contaminated pork after 6 h enrichment. A) Agarose gel electrophoresis of conventional PCR showing detection limit, B) Agarose gel electrophoresis (2%) of PSR products showing detection limit- Lane M- 100bp plus ladder, Lane 1-9: 4X106 CFU/g, 4X105 CFU/g, 4X104 CFU/g, 4X103 CFU/g, 4X102 CFU/g, 40CFU/g, 4CFU/g, 0.4CFU/g, 0.04 CFU/g, NTC – No template control and in second rowVisual detection of PSR product by addition of SYBR Green I showing detection limit- Tube 1-9: 4X106 CFU/g, 4X105 CFU/g, 4X104 CFU/g, 4X103 CFU/g, 4X102 CFU/g, 40CFU/g, 4CFU/g, 0.4CFU/g, 0.04 CFU/g, Tube 10-NTC C) Amplification curve of different dilutions of contaminated meat showing amplification till 40 CFU/ml.
Highlights Developed a PSR assay for detecting Salmonella in pork and pork products The analytical sensitivity of the PSR assay (100 fg) was 10 fold more than the conventional PCR and was comparable to real-time PCR The detection of limit of 4 CFU per gram of pork was achieved within 8 h Positive predictive value, negative predictive value and accuracy rate of the developed assay was 100%
S0890-8508(19)30401-3
DOI:
https://doi.org/10.1016/j.mcp.2020.101510
Reference:
YMCPR 101510
To appear in:
Molecular and Cellular Probes
Received Date: 15 October 2019 Revised Date:
30 December 2019
Accepted Date: 13 January 2020
Please cite this article as: Momin KM, Prince Milton AA, Ghatak S, Thomas SC, Priya GB, Das S, Shakuntala I, Sanjukta R, Puro K-u, Sen A, Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect Salmonella in pork and pork products, Molecular and Cellular Probes (2020), doi: https://doi.org/10.1016/j.mcp.2020.101510. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Contribution of each author K.M. Momin, Project staff, DST PSR
A.A.P. Milton
S. Ghatak
S.C. Thomas
G.B. Priya, SRF, DBT AMR S. Das I. Shakuntala R.K. Sanjukta K. Puro A. Sen
Performed all the major experiments (Analytical sensitivity, LOD, Field applicability, Comparison assays) Conceptualized the work, designed the experiment plan, monitored all the experiments, analyzed results, drafted the manuscript Performed specificity experiment, provided critical inputs then and there required, drafted the manuscript Designed the PSR primers used in the study. Helped in incorporation of olignonucleotide sequences of exogenous (botanic) origin with PSR primers. Performed artificial spiking experiment in pork Analyzed the results and shared reference TB cultures. Helped in designing the experiment and gave critical inputs Helped in analyzing results and setting all the figures Helped in drafting the manuscript and gave critical inputs Helped in drafting the manuscript and proof reading of the manuscript
1 2 3 4 5 6 7 8 9 10 11
Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect Salmonella in pork and pork products Kasanchi M. Momin1,§, Arockiasamy Arun Prince Milton1,§,*, Sandeep Ghatak1,*, Shiny C. Thomas2, Govindarajan Bhuvana Priya1, Samir Das1, Ingudam Shakuntala1, Rajkumari Sanjukta1, Kekungu-u Puro1, Arnab Sen1 1
Division of Animal Health, Indian Council of Agricultural Research (ICAR) Research Complex for NEH Region, Umiam, Meghalaya 793103, India 2 School of Life Sciences, Assam Don Bosco University, Guwahati, Assam, India
12 13
§: Both authors contributed equally to this work *Corresponding Authors: Dr. A.A.P Milton, Dr. S. Ghatak, Division of Animal Health,
14
ICAR Research Complex for NEH Region, Umiam- 793 103, Meghalya.
15
Email: [email protected]; [email protected]
16
Mobile: +91 8650918630; +919436380401
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32
Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect
33
Salmonella in pork and pork products
34
Abstract
35
The polymerase spiral reaction (PSR), a novel isothermal method for targeted DNA
36
amplification, was effectively applied to detect Salmonella in artificially spiked pork. The
37
specificity of the developed PSR was tested using 16 Salmonella and 15 non-Salmonella
38
strains. The PSR assay was 10-fold more sensitive than conventional end-point PCR, having
39
a sensitivity comparable to real-time PCR. The limit of detection of the developed assay was
40
4 X 103 per gram of pork without enrichment and 4 CFU per gram after a 6 h enrichment. The
41
detection of 4 CFU per gram of pork was achieved within 8 h. The PSR assay was successful,
42
and accurate in comparison to microbiological methods, in detecting Salmonella in 11 of 76
43
commercial pork samples. Therefore the positive predictive value, negative predictive value
44
and accuracy rate of the developed assay were 100%. Considering its rapidity, user-
45
friendliness, simplicity, cost-effectiveness and equipment-free nature, this PSR assay is a
46
promising tool for the food industry for the detection of Salmonella and prevention of
47
Salmonella outbreaks and recalls.
48
Keywords: Salmonella; Polymerase spiral reaction; Pork; Pathogen detection
49
1. Introduction
50
Salmonella is an important foodborne pathogen and the primary cause of bacterial
51
foodborne illnesses worldwide [1]. It causes bacterial gastroenteritis and is responsible for
52
approximately 30% of food poisoning cases in the USA (1.4 million human cases annually)
53
[2]. Globally, non-typhoidal Salmonella account for approximately 153 million cases
54
(approx) of gastroenteritis and 57,000 deaths each year [3]. The most common sources of
55
Salmonella infection are foods of animal origin. Contaminated food products such as eggs,
56
poultry, pork and beef contribute to 75 % of human Salmonella cases [4]. While data on the
57
prevalence of Salmonella spp. in foods of animal origin in India is patchy, several reports
58
indicate the public health importance of the organism [3, 5-7].
59
In healthy humans, the infectious dose is typically ≥105 Salmonella cells, but in
60
extremely susceptible individuals, as little as 15-20 cells can cause disease [8]. When present,
61
foodborne pathogens are often at very low numbers relative to background microflora,
62
making their detection and identification difficult [9]. Conventional detection methods for
63
Salmonella spp. include pre-enrichment, selective enrichment and plating on selective media
64
followed by biochemical and serological testing of presumptive isolates. While these methods
65
are highly specific and are considered as the gold standard, they are time-consuming
66
(requiring 7-10 days) and technically demanding (requiring well-trained manpower) [10]. In
67
spite of the many advantages of nucleic acid based detection methods such as PCR, and real-
68
time PCR, these methods impose many other constraints that prevent their routine use in
69
resource-limited settings (e.g., costly equipment and laboratory set-up, post-PCR processing,
70
technical training, etc.) [11]. Most of these detection methods are not appropriate for onsite
71
detection or small scale food establishments. Therefore, rapid, cost-effective, simple,
72
sensitive, specific and field portable assays are crucial for the detection of foodborne
73
pathogens to control the distribution of contaminated foods [12].
74
In recent years, many isothermal nucleic acid amplification-based assays, have been
75
developed and deployed to detect foodborne pathogens. These methods can synthesize a large
76
amount of DNA rapidly with high specificity. These assays obviate the necessity for a
77
thermocycler, gel electrophoresis unit and trained personnel. Among them, loop-mediated
78
isothermal amplification (LAMP) has been broadly applied and has been demonstrated to be
79
a powerful tool for the detection of Salmonella in foods [11-19]. Invasin A gene (invA) is the
80
most common target for nucleic acid based detection of Salmonella. The invA gene is
81
chromosomally located and encodes for an inner membrane protein of Salmonella that is
82
essential for the invasion of epithelial cells and is conserved in all the Salmonella serotypes
83
[20]. Despite the many advantages of LAMP, the technique has some important limitations
84
(the requirement for multiple primers, requiring rigorous optimization and the possibility of
85
carryover or leftover contamination leading to false-positive results) that have limited its
86
adoption [21].
87
Polymerase Spiral Reaction (PSR) is a novel isothermal assay which rapidly amplifies
88
target nucleic acid in the temperature range of 60–650C with high sensitivity and specificity.
89
In contrast to LAMP, it requires only two primers [22]. The technique does not require any
90
sophisticated instruments and has successfully been demonstrated as a potential point-of-care
91
test for detecting many pathogens of medical and veterinary significance [23-28]. However, it
92
has seldom been applied for the detection of foodborne pathogens and has yet to be adopted
93
in the food industry. More recently, a PSR assay for detection of Salmonella in food was
94
standardized [29]. However the main limitations of the study are that they have used two pair
95
of primers, artificial spiking experiment was not performed to determine the limit of detection
96
(LOD) and the effect of enrichment in improving the LOD was not studied.
97
Therefore, the present study aimed to develop and evaluate a PSR assay targeting the
98
invA gene to detect Salmonella in foods. The developed assay was validated by detecting
99
Salmonella in artificially contaminated pork. The effect of a short culture enrichment on
100
detection limit of Salmonella by PSR was also studied. The sensitivity and specificity of the
101
developed PSR were also compared to PCR and real-time PCR methods. To avoid primer
102
bias, PSR assay was compared with newly optimised PCR and real-time PCR assays
103
targeting same conserved region of invA gene of Salmonella. We chose to use same primers
104
(except the botanical portion of the PSR primers) in all three assays (PCR, real-time PCR and
105
PSR) as our aim was to compare assays, not primers. However analytical sensitivity of PCR
106
and real-time PCR assays based on well-cited primers were also compared with the current
107
PSR assay. The portability of the developed assay was also demonstrated by screening field
108
pork samples.
109
2. Materials and Methods
110
2.1 Bacterial strains
111
Five Salmonella reference strains, 11 Salmonella strains isolated from food samples
112
and 15 non-Salmonella strains (Table 1) were used in the present study. The strains were
113
obtained from American Type Culture Collection (Manassas, Virginia, USA) and the
114
National Salmonella Centre (Indian Veterinary Research Institute, Bareilly, Uttar Pradesh,
115
India). Bacterial strains were stored as 20% glycerol stocks at -80°C. Strains were streaked
116
on suitable media (HiMedia, India) and grown at 37 °C. Broth cultures were inoculated from
117
single colonies into 10 mL of appropriate media (HiMedia, India) and grown overnight at 37
118
°C.
119
Typhimurium ATCC 51812 was used as a reference strain in the analytical sensitivity and
120
meat spiking studies.
121
Table 1. Bacterial strains used in the study
Cells were harvested and used to prepare DNA as described below. Salmonella
Bacterial Species
Salmonella Strains Salmonella Typhimurium Salmonella Enteritidis Salmonella Paratyphi Salmonella Pullonum Salmonella Uccle Salmonella food isolates (n=11) non-Salmonella Strains Shigella boydii Shigella sonnei Escherichia coli Enterococcus faecalis Pseudomonas aeruginosa
Strain/ Source*
ATCC 51812 NSC 2478 NSC 77 NSC E79 NSC 60a
ATCC 25931 ATCC 9207 ATCC 25922 ATCC 51299 ATCC 10145
122
Listeria monocytogenes ATCC 13119 Klebsiella pneumonia ATCC 700608 Klebsiellaoxytoca ATCC 43863 Campylobacter jejuni ATCC 33291 Staphylococcus aureus ATCC 33591 Staphylococcus xylosus ATCC 29971 Staphylococcus epidermidis ATCC 12228 Clostridium perfringens ATCC 13124 Mycobacterium smegmatis ATCC 607 Mycobacterium bovis AN5 *ATCC-American Type Culture Collection (USA); NSC-National Salmonella Centre (India)
123
2.2 Extraction of genomic DNA
124
Genomic DNA of both Salmonella and non-Salmonella strains was isolated using
125
QIAmp DNA Mini Kit (Qiagen, Germany) as per manufacturer’s instructions. The
126
concentration of the extracted DNA were measured using UV-Visible spectrophotometry
127
(Nanodrop,Thermo Scientific, USA). The DNA preparations were stored at -20 °C.
128
2.3 Designing of PSR primers
129
The PSR primers were designed to target the conserved region of the invA gene (Gen
130
Bank Accession number CP043222) of Salmonella using the Primer3 program (NCBI). The
131
principles of PSR primer design were described previously [22]. The Primer3 suggested
132
primers
133
(https://blast.ncbi.nlm.nih.gov/Blast.cgi). The selected primers were synthesized by Imperial
134
Life Scienes (Haryana, India). The in-house PCR and real-time PCR targeting the invA gene
135
were used to compare the sensitivity and specificity of developed PSR assay (data not
136
shown). The primer sequences designed and used for PSR, PCR and realtime PCR are given
137
in Table 2.
138
Table 2. Primer sequences used in the study
Assay
were
checked
for
specificity
in
silico
with
the
BLAST-N
Primer
Sequences
INV2PF
5’-acgattcgtacatagaagtatagTGGATTTGTCCTCCGCCCTG-3’
program
Product size Variable
Source This study
PSR PCR & realtime PCR
INV2PR
5’-gatatgaagatacatgcttagcaCCGTATCGCCATTTACGCGG-3’
INV2F
5’-TGGATTTGTCCTCCGCCCTG-3’
INV2R
5’-CCGTATCGCCATTTACGCGG-3’
This study This study 129 bp This study
139 140
2.4 Optimization of the PSR and visual detection of amplified products
141
The PSR assay was optimized for assay temperature (60-70 °C) and time (15-90 min)
142
as well as dNTP (0.5-1.6 mM) (Thermoscientific, USA), MgSO4 (2.0-8.0 mM) (New England
143
BioLabs, USA), betaine (0.5-1.4mM) (Sigma-Aldrich, USA), BST 2.0 Warm start
144
polymerase (4-12 U) (New England BioLabs, USA) and primer (5µM-15µM)concentrations.
145
PCR and real-time PCR assays were optimized to compare the PSR assay. The species-
146
specific primer sets for Salmonella enterica were designed from the same conserved region
147
of invA gene chosen for PSR (Table 2). The PCR reaction mixture (total volume, 25 µl)
148
contained 12.5 µl of 2x Dream Taq Master Mix (Dream Taq DNA polymerase, 2X Dream
149
Taq Green buffer, 0.4mM dNTPs, 4mM MgCl2), 1µl of 10pmol each of forward and reverse
150
primer, 1 µl of the template DNA and 8.5µl nuclease-free water. Amplification was
151
performed in Mastercycler nexus GX2 (Eppendorf, Germany). The cycling conditions
152
consisted of 10 min initial denaturation at 94 °C, followed by 35 cycles each of 1min
153
denaturation at 94 °C, 1.5 min annealing at 62 °C and 1min elongation at 72 °C and a final
154
extension step of 5 min at 72 °C. Real-time PCR was carried out in a 20µl reaction volume
155
consisting of 10µl Power SYBR Green PCR Master Mix (Applied Biosystems), 0.1µl each of
156
forward and reverse primer, 7.8µl of nuclease-free water and 1µl sample DNA. The reaction
157
was carried out in 7500 Fast Real-Time PCR System (Applied Biosystems) in following
158
conditions: initial denaturation at 95 °C for 10 minutes, followed by 40 cycles each of 15 sec
159
denaturation at 94 °C, 1 min annealing at 60 °C and 1min elongation at 72 °C and a final
160
extension step of 5 min at 72 °C. The real-time PCR conditions consisted of an initial step of
161
95°C for 10 minutes followed by an amplification program for 40 cycles of 15 seconds at
162
95°C, 1 minute at 60°C, 1 minute at 72°C with fluorescence acquisition at the end of each
163
extension. The amplification program was immediately followed by a melt program
164
consisting of 15 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 95°C with
165
fluorescence acquisition at each temperature transition. The PCR amplicon was detected by
166
electrophoresis on 1.5% agarose gel stained with ethidium bromide (0.5 µg/ml) and
167
photographed using gel imaging system (AlphaImager, UK). The amplified PSR products
168
were detected visually (under white light) by the addition of 1 µl of SYBR Green I dye
169
(10,000 X, Sigma-Aldrich, USA) diluted 1:10 in 1X phosphate buffered saline (PBS) which
170
produced fluorescent green colour in positive whereas the colour remained unchanged
171
(orange colour) in the negative reactions. The PSR products were also subjected to agarose
172
gel electrophoresis similar to PCR products and photographed for confirmation.
173 174
2.5 Analytical sensitivity and specificity
175
To determine the analytical sensitivity, serial tenfold dilutions (10-1 to 10-8) of the
176
extracted genomic DNA of Salmonella Typhimurium ATCC 51812 were prepared and used
177
to perform PSR, PCR and real-time PCR as per the standardized protocols. In real-time PCR,
178
a Ct value of 32 was set as a cut-off point. Analytical sensitivity of the PSR assay was also
179
compared with the previously published invA based PCR [30] and real-time PCR assays [31].
180
The specificity of the developed PSR assay was determined by checking for its cross-
181
reactivity with genomic DNA of 16 Salmonella and 15 non-Salmonella species/strains of 10
182
different genera.
183
2.6 Artificial contamination study and limit of detection (LOD) of the PSR assay
184
The limit of detection (LOD) of the PSR assay was estimated using artificially
185
contaminated pork as a model meat. A single S. Typhimurium (ATCC 51812) colony was
186
picked from an XLD agar (HiMedia, India) plate and was cultured in 10 ml of tryptone soy
187
broth (HiMedia, India) at 37 °C overnight (16 hr). After the overnight incubation, the
188
Salmonella cells were harvested by centrifugation at 10,000 x g for 10 min. The bacterial
189
pellet was washed once with 2 ml of PBS and re-suspended in final volume of 2 ml PBS.
190
Serial 10 fold dilutions (10-1 to 10-8) of this bacterial suspension were made in 1X sterile
191
PBS. The bacterial concentration in overnight culture was deteremined by spreading 100 µl
192
of serially diluted suspensions onto XLD agar plates in duplicate followed by overnight (18
193
h) incubation at 37 °C. The pork cut was purchased from the local market in Umiam,
194
Meghalaya. The pork was confirmed to be free from Salmonella by real-time PCR and a
195
microbiological culture method (ISO 6579:2002; [32]). Pork homogenate was prepared by
196
stomaching 25 g of meat in 225 ml of 0.1% buffered peptone water (HiMedia, India) and 9
197
ml of the homogenates were distributed in 15 ml culture tubes. One ml of each dilution of the
198
prepared bacterial suspension was inoculated into 9 ml of the pork homogenate dispensed in
199
15 ml culture tubes. A negative control was created by mixing 9 ml of the meat homogenate
200
with 1 ml sterile 1X PBS. Genomic DNA was extracted using the DNeasy blood and tissue
201
kit (Qiagen, Germany) following the manufacturer’s protocol. Conventional PCR, real-time
202
PCR and PSR assays were performed using extracted DNA samples.
203
2.7 Evaluation of the effect of enrichment on LOD of the PSR assay
204
To evaluate the effect of enrichment, samples from each dilution of inoculated pork
205
were collected after 6h of incubation at 37 °C. DNA was extracted from enriched samples.
206
Briefly, 2 ml of the sample was collected from each dilution of spiked pork homogenate at 0
207
h (without enrichment) and after 6 h of enrichment and centrifuged at 10,000 g for 5 min. The
208
resulting cell pellet was then re-suspended in 180 µl of lysis buffer and was processed for
209
genomic DNA extraction as per manufacturer’s protocol (Qiagen, Germany). Extracted DNA
210
was used as the template in PCR, real-time PCR and PSR assays and the LOD were
211
compared between the assays. All experiments using artificially contaminated pork were
212
done in triplicate.
213
2.8. Application of the developed PSR assay to field samples
214
To evaluate applicability of the developed PSR assay to naturally contaminated samples,
215
it was deployed to screen raw pork and processed pork samples (n=76) for the presence of
216
Salmonella from the retail market of Meghalaya. Samples were obtained from the local wet
217
markets of Meghalaya. The accuracy of the developed PSR assay was compared with the
218
cultural methods, conventional PCR and real-time PCR. Salmonella isolation and
219
identification was done according to ISO 6579:2002 methods [32]. Additional confirmation
220
of all isolates was done by PCR targeting the invA gene.
221
2.9 Statistical analysis
222
The performances of the newly developed diagnostic PSR, PCR and real-time PCR
223
assays were evaluated by determining the sensitivity, specificity, Positive Predictive Value
224
(PPV), Negative Predictive Value (NPV) and Accuracy. Calculations were based on the final
225
detection for each Salmonella isolate tested. True positive and true negative samples were
226
based on the results of Salmonella isolation and identification employing the ISO 6579:2002
227
method. PPV, NPV and accuracy were calculated by the following formulae
228 229 230 231 232 233 234 235 236 237 238 239
PPV=
(Number of TP) (Number of TP + Number of FP)
x 100
NPV=
(Number of TN) (Number of TN + Number of FN)
x 100
Accuracy=
(Number of TP + TN) (Number of TP + FP + TN + FN)
x 100
Where - TP: true positive, FP: false positive, TN: true negative, FN: false negative
240
3. Results
241
3.1 Optimization of the PSR assay
242
PSR was successfully optimized for all the reagents, temperature and time (Figures
243
1A-G). The optimized PSR mixture contained 2.5 µl of 10X Isothermal amplification buffer
244
(New England BioLabs, USA), 8.0 mM MgSO4, 1.4 mM dNTP’s, 0.8 M betaine,8.0 U Bst
245
2.0 WarmStart DNA polymerase, 10 µM forward primer, 10 µM reverse primer,1.0 µl of
246
template genomic DNA and nuclease-free water to make up the volume to 25 µl. The reaction
247
was incubated at 64 °C for 60 min. PSR amplicons were detected by the addition of SYBR
248
Green I dye and UV illumination. Positive samples yielded green fluorescence, whereas
249
negative reactions remained the original orange colour. The PSR amplicons were also
250
subjected to agarose gel electrophoresis which revealed a ladder pattern when stain with
251
ethidium bromide (Figures 1A-G).
252
3.2 Specificity or exclusivity of the PSR assay
253
The developed PSR assay for Salmonella targeting the invA gene demonstrated a high
254
degree of specificity, amplifying the genomic DNA of all 16 Salmonella strains tested and
255
yielding negative results with genomic DNA the other 15 bacterial species tested. The results
256
of specificity are presented in figure (Figure 2).
257
3.3 Analytical sensitivity of the PSR, PCR and real-time PCR assays
258
The analytical sensitivity of the developed PSR assay was determined and compared
259
with the results of newly developed and published PCR and real-time PCR assays. The
260
genomic DNA extracted from the pure culture of S. Typhimurium (ATCC 51812) had a
261
concentration of 100 ng/µl. The DNA concentration of ten-fold serial dilutions ranged from
262
10 ng/µl (100X10-1 ng/µl) to 1fg/µl (100X10-8 ng/µl). Since 1 µl of template DNA solution
263
was used in each amplification reaction, the total DNA per assay ranged from 10 ng to 1 fg.
264
The analytical sensitivity of newly developed PSR, real-time PCR and PCR assays were
265
found to be 100 fg, 100 fg and 1 pg, respectively indicating PSR to be 10 times more
266
sensitive than PCR and comparable to real-time PCR (Fig 3). The analytical sensitivity of
267
current PSR, previously published real-time PCR and PCR assays were found to be 100 fg, 1
268
pg and 10 pg, respectively indicating PSR to be 100 times more sensitive than previously
269
published PCR and 10 times sensitive than real-time PCR (Fig 4).
270
3.4 Artificial contamination study and limit of detection (LOD) of PSR,PCR and real-time
271
PCR assays
272
The bacterial concentration in the initial culture of S. Typhimurium ATCC 51812 was
273
4 x 107 CFU/ml. The homogenized pork (9 ml) was inoculated with 1 ml of 10-fold serially
274
diluted S. Typhimurium (ATCC 51812) culture prepared in 1X PBS. Therefore, inoculated
275
pork was carrying S. Typhimurium ranging from 4 x 106 CFU to 0.04 CFU of Salmonella in
276
the absence of culture enrichment, the detection limit of PCR, real-time PCR and PSR was
277
found to be 4 x 104 CFU/g, 4 x 103 CFU/g and 4 x 103 CFU/g of meat, respectively (Fig 5).
278
After enrichment for 6 h, the detection limit of PCR, real-time PCR and PSR was found to be
279
4 x 102 CFU/g, 40 CFU/g and 4 CFU/g of meat, respectively (Fig 6). The negative control did
280
not show any amplification, further confirming the specificity of developed PSR assay.
281
3.5 Real-world applicability of developed PSR assay
282
To evaluate real-world applicability of the developed PSR assay, it was deployed to
283
screen Salmonella from the raw, processed and cooked pork samples (n=76) collected from
284
retail markets of Meghalaya. The samples were screened with conventional PCR, real-time
285
PCR, PSR and conventional cultural methods. Out of 76 samples, following ISO 6579:2002
286
cultural method, Salmonella could be isolated from 11 pork samples. Without enrichment (at
287
0 hours), none of the samples gave positive results with conventional PCR, real-time PCR
288
and PSR assays. With a brief enrichment of 6 hours, PCR, real-time PCR and PSR could
289
detect Salmonella from 8, 11 and 11 samples, respectively.
290
3.6 Estimation of sensitivity, specificity, PPV, NPV and accuracy
291 292 293 294 295
The sensitivity, specificity, PPV, NPV and accuracy of the PSR, PCR and real-time PCR are furnished in the table 3.
296
Table 3. PPV, NPV, and accuracy (expressed as percentage, %) of PSR, PCR and real-
297
time PCR assays compared to ISO 6579:2002 Assays
298
Sensitivity Specificity PPV NPV Accuracy (95% LCL- (95% LCL- (95% LCL-95% (95% LCL- (95% LCL95% UCL) 95% UCL) UCL) 95% UCL) 95% UCL) PSR 100 100 100 100 100 (67.8-100.0) (93.0-100.0) (67.8-100.0) (93.0-100.0) (95.2-100.0) PCR 72 100 100 95.6 96.0 (39.3-92.7) (93-100.0) (59.7-100.0) (86.8-98.8) (88.9-99.1) Real-time 100 100 100 100 100 PCR (67.8-100.0) (93.0-100.0) (67.8-100.0) (93.0-100.0) (95.2-100.0) LCL, lower confidence limit; UCL, upper confidence limit
299
The PPV and NPV of the newly developed PSR assay for detection of Salmonella was 100%.
300
The accuracy of the newly developed PSR assay was also calculated to be 100%.
301
4. Discussion
302
Foodborne non-typhoidal salmonellosis remains an important economic and public health
303
burden for both developing and developed countries [3]. Most of the existing diagnostic
304
methods to detect Salmonella in foods suffer from many shortcomings, for example, they are
305
time consuming and expensive, exhibit low sensitivity, require trained manpower, yield a
306
high rate of false positivities (due to dead bacteria) etc. Considering the speed at which the
307
food moves from farm to fork and how rapidly an outbreak of human salmonellosis can
308
occur, rapid diagnostic methods are the clear priority and an urgent need from the food safety
309
and public health perspective. The rapidity in detecting the causative agent is crucial not only
310
for preventing hospitalization or clinical complication, but also for spotting the source of the
311
outbreak. This helps in efficient removal of the contaminated food products from the food
312
distribution chain. Ideally, pathogen detection would occur prior to distribution and
313
consumption of contaminated foods, reducing the occurrence of a food borne outbreaks.
314
Moreover, time consuming techniques such as conventional cultural methods (ISO 6579:
315
2002) [32] are not suitable for foods with a short shelf life like meat or ready to eat foods
316
[33]. In developing countries, the food regulatory agencies usually do not have sophisticated
317
laboratories, therefore simple techniques without the need for costly equipment and trained
318
manpower is a vital requirement.
319
Considering all this, we have developed a simple, rapid and promising polymerase spiral
320
reaction (PSR) assay to detect Salmonella in foods of animal origin. Moreover, we
321
demonstrated the potential of the PSR assay in rapid and accurate detection (<8 hours) of
322
Salmonella from raw pork and pork products. LAMP assays are frequently touted as simple
323
and rapid nucleic acid-based method for the detection of pathogens [8]. Furthermore, several
324
LAMP-based assays have been developed for detecting Salmonella from different foods of
325
animal origin [12, 14, 15, 34, 35]. The novelty of the present PSR assay in comparison with
326
LAMP assays is the requirement of only a single pair of primers that simplifies assay
327
development and eliminates the demand of rigorous optimization. Compared to LAMP, the
328
need for only two primers in PSR minimizes the likelihood of non-specific amplification and
329
contamination [27]. Thus, PSR is a unique blend of an isothermal amplification technique
330
and conventional PCR (single pair of primer).
331
The present PSR assay to detect Salmonella proved to be superior to conventional PCR as
332
it does not require a thermocycler, reducing the need for post PCR processing and has higher
333
sensitivity. Bst polymerase used in PSR and LAMP is shown to be more resistant to the
334
inhibitors of Taq DNA polymerase used in conventional PCR [36]. In the present study, the
335
analytical sensitivity of PSR and conventional PCR were 100 femtograms and 1 picogram,
336
respectively. The limits of detection in artificially contaminated pork without culture
337
enrichment were 4 x 104 CFU/g and 4 x 103 CFU/g in PCR and PSR, respectively. This
338
indicates that the present PSR assay was 10 fold more sensitive than the PCR (without
339
enrichment). After enrichment for 6 h, the detection limits of PCR and PSR were found to be
340
4 x 103 CFU/g, and 40 CFU/g of meat, respectively. So after a brief enrichment of 6 hours,
341
the PSR performed 100 times better than the conventional PCR assay.
342
The developed PSR assay also has the advantage over real-time PCR in that PSR does not
343
required costly and bulky equipment for post-PCR analysis. Moreover, the present PSR
344
assay was found to have comparable analytical sensitivity with that of real-time PCR (100
345
femtograms of genomic DNA). Furthermore, in artificially contaminated pork without
346
enrichment, both the PSR and real-time PCR had the same detection limits (4 x 103 CFU/g of
347
meat) and, with a brief enrichment of 6 h, the PSR assay (40 CFU/g of meat) was 10 times
348
more sensitive than the real-time PCR assay (400 CFU/g of meat).
349
The analytical sensitivity of 100 femtograms in the present assay is in concordance with
350
the earlier EMA (ethidium monoazide) LAMP and standard LAMP assays developed by Lu
351
et al. [34] and Wang et al. [14], respectively for detection of Salmonella in raw chicken and
352
pork. The limit of detection of the present PSR assay was found to be better than the earlier
353
RT-LAMP assay developed by Techathuvanan et al [12] for the detection of Salmonella from
354
pork products. They have reported a detection limit of 102CFU/ 25 g after enrichment and 106
355
CFU/25 g without enrichment. The detection efficiency of our assay is also better than an
356
earlier RT-LAMP assay developed for pork processing environment samples, wherein a limit
357
of detection of 10 CFU/ml was reported [37]. The current assay was also found to be more
358
efficient and rapid than an earlier assay which reported a detection limit of 35 CFU of
359
Salmonella per 250 ml of minced pork and raw milk with the assay time of 24 h that
360
includedtime for sample enrichment [15]. Our assay is also more sensitive than a real-time
361
LAMP assay developed for detection of Salmonella which has detected 7 CFU/ml in the
362
artificially spiked chicken samples after enrichment of 6 h [35] that is only slightly less
363
sensitive than our detection limit of 4 CFU/g after a 6 h enrichment.
364
In recent years, few PSR assays have been developed to detect important pathogens.
365
Among them, most of the assays have revealed a 10 fold higher sensitivity than conventional
366
PCR assay [23-26], which is in line with our result. However, PSR assays to detect bovine
367
herpes virus [26], Brucella [27] and Mycoplasma synoviae [28] have shown 100 times greater
368
sensitivity than conventional PCR. Similar to our PSR assay, previously reported assays have
369
shown comparable sensitivity to real-time PCR [25, 27, 29].
370
In the evaluation of the developed PSR assay for its field applicability, naturally
371
contaminated or field pork samples were tested with conventional PCR, real-time PCR, PSR
372
and conventional cultural methods. The conventional cultural method revealed 11
373
Salmonella-positive samples from 76 samples tested. None of the other assays could detect
374
Salmonella when employed in unenriched samples. However, after enrichment for 6 h, PCR,
375
real-time PCR and PSR could detect Salmonella from 8, 11 and 11 samples, respectively.
376
Although the limit of detection of the PSR assay before enrichment in the artificial
377
contamination study was 4 x 103 CFU/g of pork, this negative result might be due to lower
378
levels of contamination (<4 x 103 CFU/g)in the field samples. As the initial count of
379
Salmonella in raw foods samples are usually low [38], it is suggested that the present PSR
380
assay be employed after a 6 h enrichment. This also ensures the detection of viable
381
Salmonella from the samples. Finally, the complete operation time of the developed PSR
382
assay including DNA extraction and the enrichment step can be completed in approximately
383
8 hour.
384
The main advantage of this study is that a novel, simple and rapid assay was developed to
385
detect Salmonella in pork and pork products in resource-limited settings. Our study evaluated
386
the effect of enrichment and compared the developed assay with conventional PCR and real-
387
time PCR assays. The field applicability was also evaluated by screening naturally
388
contaminated or field samples and comparison was done with the gold standard cultural
389
method(ISO 6579:2002) [32]. While the developed PSR assay gives accurate results in both
390
pork and pork products, additional studies will be necessary to extend its application to other
391
foods.
392
In conclusion, we successfully developed and evaluated novel PSR assay for detection
393
of Salmonella from foods, which complies with the “ASSURED (affordable, sensitive,
394
specific, user-friendly, robust and rapid, equipment-free, and deliverable)” concept proposed
395
by WHO for the development of diagnostic. Since our assay has multiple advantages over
396
many Salmonella diagnostic assays currently employed; we foresee that it has the potential to
397
become the assay of choice for routine detection of Salmonella in small or resource-limited
398
food testing laboratories.
399 400
Competing Interests
401
The authors declare that there are no competing interests
402
Acknowledgements
403
The authors are thankful to the Department of Science and Technology (DST),
404
Ministry of Science and Technology, New Delhi, Government of India, for sanctioning the
405
project (research grant no. SP/YO/570/2018(G)). The authors are also grateful to the
406
Director, ICAR Research Complex for NEH Region, Umiam, Meghalaya, India for providing
407
necessary facilities to conduct this research work. We sincerely acknowledge the contribution
408
of Dr George C Paoli, Research Microbiologist, USDA ARS in improving the manuscript.
409 410
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411
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Fig. 1. (A-G). Optimization of reagents (A. Primer; B. dNTP; C. MgSo4; D. Betaine; E. Bst Polymerase; F. Temperature; G. Time) concentration for PSR assay. First row- Visual detection with SYBR Green I showing green fluorescence in amplified products and orange in tubes with no amplification. Second row- Electrophoretic pattern of PSR amplified products. (Lane M- 100 bp plus ladder; NTC- Non-template control) Fig. 2 Specificity of PSR assay. First row- Agarose gel electrophoresis of PSR products (Lane 1-15) showing no amplification in non- Salmonella DNA and amplification in Salmonella DNA (Lane 16-20). Second row- Visual detection of PSR products (corresponding tube numbers 1-15) showing no amplification in non- Salmonella DNA and showing amplification in Salmonella DNA (corresponding tube numbers 16-20) using SYBR Green dye. (Lane M- 100 bp plus ladder; NTC- Non-template control) Fig. 3. Analytical sensitivity A) Agarose gel electrophoresis of new conventional PCR showing analytical sensitivity, B) Agarose gel electrophoresis (2%) and visual detection of PSR products showing analytical sensitivity, (Lane M- 100bp plus ladder, NTC- Nontemplate control) C) Amplification curve showing analytical sensitivity of new real-time PCR Fig. 4. Analytical sensitivity A) Agarose gel electrophoresis of conventional PCR (previously published [30]) showing analytical sensitivity (Lane M- 100bp plus ladder, NTC- Nontemplate control), B) Amplification curve showing analytical sensitivity of real-time PCR (previously published [31]). Fig. 5. Limit of detection in artificially contaminated pork (at 0 hour). A) Agarose gel electrophoresis of conventional PCR showing limit of detection, B) Agarose gel electrophoresis (2%) of PSR products showing limit of detection (Lane 1-9: 4X106 CFU/g, 4X105 CFU/g, 4X104 CFU/g, 4X103 CFU/g, 4X102 CFU/g, 40CFU/g, 4CFU/g, 0.4CFU/g, 0.04 CFU/g, NTC – No template control) and in second row- Visual detection of PSR products by addition of SYBR Green I showing limit of detection (Tube 1-9: 4X106 CFU/g, 4X105 CFU/g, 4X104 CFU/g, 4X103 CFU/g, 4X102 CFU/g, 40CFU/g, 4CFU/g, 0.4CFU/g, 0.04 CFU/g, Tube 10- NTC); C) Amplification curve of different dilutions of contaminated meat showing amplification till 4X103CFU/g. Fig. 6. Limit of detection in artificially contaminated pork after 6 h enrichment. A) Agarose gel electrophoresis of conventional PCR showing detection limit, B) Agarose gel electrophoresis (2%) of PSR products showing detection limit- Lane M- 100bp plus ladder, Lane 1-9: 4X106 CFU/g, 4X105 CFU/g, 4X104 CFU/g, 4X103 CFU/g, 4X102 CFU/g, 40CFU/g, 4CFU/g, 0.4CFU/g, 0.04 CFU/g, NTC – No template control and in second rowVisual detection of PSR product by addition of SYBR Green I showing detection limit- Tube 1-9: 4X106 CFU/g, 4X105 CFU/g, 4X104 CFU/g, 4X103 CFU/g, 4X102 CFU/g, 40CFU/g, 4CFU/g, 0.4CFU/g, 0.04 CFU/g, Tube 10-NTC C) Amplification curve of different dilutions of contaminated meat showing amplification till 40 CFU/ml.
Highlights Developed a PSR assay for detecting Salmonella in pork and pork products The analytical sensitivity of the PSR assay (100 fg) was 10 fold more than the conventional PCR and was comparable to real-time PCR The detection of limit of 4 CFU per gram of pork was achieved within 8 h Positive predictive value, negative predictive value and accuracy rate of the developed assay was 100%