A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis

Accepted Manuscript A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis Aqeela Ashraf, Muhammad Imran, Tahir Yaqub, Muhammad Tayyab, Wasim Shehzad, Peter C. Thomson PII:

S0890-8508(17)30023-3

DOI:

10.1016/j.mcp.2017.03.004

Reference:

YMCPR 1279

To appear in:

Molecular and Cellular Probes

Received Date: 28 November 2016 Revised Date:

18 March 2017

Accepted Date: 19 March 2017

Please cite this article as: Ashraf A, Imran M, Yaqub T, Tayyab M, Shehzad W, Thomson PC, A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis, Molecular and Cellular Probes (2017), doi: 10.1016/j.mcp.2017.03.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

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Title

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A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial

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pathogens associated with bovine mastitis.

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Authors

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1. Aqeela Ashrafa

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2. Muhammad Imrana (Corresponding author)

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3. Tahir Yaqubb

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4. Muhammad Tayyaba

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5. Wasim Shehzada

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6. Peter C. Thomsonc

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a

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Shaykh Abdul Qadir Jilani Road, Lahore 54000, Pakistan.

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b

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Qadir Jilani Road, Lahore 54000, Pakistan.

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c

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Camden, 2570, NSW, Australia & Adjunct Professor, University of Veterinary and Animal

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Sciences, Shaykh Abdul Qadir Jilani Road, Lahore 54000, Pakistan.

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ABSTRACT

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For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific

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multiplex PCR assay was developed. The assay was standardized using reference strains and

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validated on mastitic milk cultures which were identified to species level based on 16S rRNA

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sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly

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from milk. Detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and

Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences,

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Department of Microbiology, University of Veterinary and Animal Sciences, Shaykh Abdul

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School of Life and Environmental Sciences, The University of Sydney, 425 Werombi Road,

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104 CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was

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sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the

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bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could

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be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological

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safety of milk, and conducting epidemiological studies.

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Keywords:

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Bovine mastitis, multiplex PCR assay, latent class analysis

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1. Introduction

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Bovine mastitis is the most common and significant disease of dairy animals. It is multi-factorial

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in nature and very difficult to control due to the involvement of a large number of pathogens.

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According to surveys, mastitis is one of the major diseases in the Pakistan dairy sector [1]. The

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extensive knowledge of the etiology of mastitis is fundamental for the development of an

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efficient diagnostic technique as well as control of the disease. More than 150 bacterial species

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are identified as mastitic pathogens. There are three major categories of bacteria that can infect

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the bovine mammary gland. They are environmental, contagious and the opportunist microbes

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[2]. The contagious pathogens live on the udder and are transmitted from infected to uninfected

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teats during the milking process. They mainly include; Streptococcus agalactiae (S. agalactiae),

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Staphylococcus aureus (S. aureus) and Mycoplasma bovis (M. bovis). Environmental pathogens

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usually reside in the housing and bedding and tend to enter the teat canal during the milking

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process; most common among them are Streptococcus uberis (S. uberis) and Streptococcus

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dysagalactiae (S. dysagalactiae) and environmental coliforms (gram negative bacteria

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Escherichia coli (E. coli), Klebsiella spp., Citrobacter spp., Enterobacter spp., (including

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Enterobacter faecalis and Enterobacter faecium), and other gram negative bacteria such as

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Serratia, Pseudomonas and Proteus [3]. Coagulase negative species Staphylococcus epidermidis

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(S. epidermidis), Staphylococcus simulans, Staphylococcus saprophyticus, and Staphylococcus

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chromogenes (S. chromogenes) are the opportunist pathogens and they stay on the lining of teat

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or udder skin [4].

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The primary diagnosis for mastitis is based on physiological symptoms visible to naked eye,

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such as swelling and inflammation of the mammary gland or the apparent changes in the milk.

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Because such symptoms appear only at the chronic or clinical state of mastitis, its earlier

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diagnosis relies on a multitude of simple diagnostic methods. The most commonly employed

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among these methods are the measurement of somatic cell count and enzymatic analysis [5].

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California mastitis test and Surf field mastitis test are also used. But these simple methods hold

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many discrepancies which could increase the likelihood for false positive or false negative

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findings. Also these simple methods are unable to provide information on the identity of the

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causative agent and severity of the infection. Accurate information on these factors is

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prerequisite to tailor an effective treatment, which is routinely obtained by culturing methods.

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The bacterial culture is however very expensive, time-consuming and labor-intensive [6].

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There has been remarkable progress in molecular biology-based techniques in the last few years.

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Molecular diagnostics have the ability to identify the organism with great sensitivity and

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specificity and can also distinguish between very closely related organisms. These molecular

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diagnostic methods have many advantages over the traditional bacteriology techniques in terms

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of low cost and accurate detection. With the advancement in molecular techniques, quick and

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accurate diagnosis of veterinary diseases has become possible [7]. PCR is a promising tool for

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efficient and accurate identification of microbes. It has become the routine diagnostic method for

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diagnosis of various plant and animal diseases, despite false positive results can be obtained even

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after the underlying infection has been cured [8].

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The performance of diagnostic tests is usually determined by sensitivity (proportion of true

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positive among diseased) and specificity (the proportion of true negative among non-diseased).

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The evaluation of a diagnostic test is therefore dependent on a reference population, the

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population of truly infected and the population of truly non-infected subjects [9]. However, no

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perfect reference or gold standard for the diagnosis of mastitis is available [10]. One of the

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frequently used approaches to evaluate a diagnostic test with unknown infection status when

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multiple tests are available is latent class analysis (LCA) [11]. LCA still assumes infection status

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is dichotomous (animal is either positive or negative) and such a status does not need to be

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known as it is assumed to be latent. This Bayesian approach has been frequently used to evaluate

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various diagnostic assays of veterinary concern [12].

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The current study was designed to develop a multiplex PCR assay for the detection of nine

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critically important bacterial pathogens associated with bovine mastitis and compare the LCA

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sensitivity and specificity estimates of the developed assay with those of bacterial culture and

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16S rRNA sequence analysis.

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2. Materials and Methods

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2.1. Bacterial strains

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The bacterial strains used for the assay development were obtained through the American Type

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Culture Collection (Manassas, VA) and Quality Milk Production Services (Cornell University,

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Ithaca, NY). These reference strains included S. agalactiae, S. dysagalactiae, S. uberis, S.

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aureus, E. coli, Staphylococcus haemolyticus (S. haemolyticus), S. chromogenes, M. bovis and S.

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epidermidis. All of these bacterial isolates were grown at 37°C on sheep blood agar, with further

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growth enrichment in a nutrient broth. For the growth of M. bovis, Hayflick agar plates

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containing 15% horse serum were incubated at 35oC with CO2 enrichment for 48 hrs or till

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colonies appeared.

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2.2. Extraction of bacterial genomic DNA

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DNA was extracted from bacterial cultures using Qiagen DNeasy Blood and Tissue Kit (Life

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Technologies, Carlsbad, CA). Genomic DNA was quantified using Nanodrop 2000

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spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and stored at -20oC until

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further use.

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2.3. Primer designing

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Multiplex PCR assay primers were designed from intraspecific conserved regions of target

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genes. The primers were designed using Primer3 software v0.4.0 [13] and synthesized through

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services provided by Invitrogen (Minneapolis, MN, USA). Detailed information on targeted

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organisms and genes, primer sequences and size of PCR amplicons is presented in Table 1.

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Table 1. Oligonucleotide primers used for multiplex PCR assay Organism M. bovis

S. dysagalactiae

S. uberis

Primer name Mb-F

Primer sequence

rRNA

Mb-R

TTGAGCCCCAAAATTTAACG

16S

Sg-F

CGCTGAGGTTTGGTGTTTACA

rRNA

Sg-R

CACTCCTACCAACGTTCTTC

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S. agalactiae

Target gene 16S

S. aureus

E. coli

S. haemolyticus

GATGTTTAGCGGGGTTGAGA

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16S

Sd-F

ACCATGTGACGGTAACTAACCA

rRNA

Sd-R

TATTACCGGCAGTCTCGCT

Cpn60

Su-F

AATTGGCATTCGTCGCGGTA

Su-R

GCATCCCTTCAACCACTTCAA

16S

Sa-F

GAACCGCATGGTTCAAAAGT

rRNA

Sa-R

CATTTCACCGCTACACATGG

phoA

Ec-F

ACGAAAAAGATCACCCAACG

Ec-R

GATCCTTTTCCGCCTTTTTC

Sh-F

AGTCGAGCGAACAGACAAGG

16S

5

Amplicon size (bps) 354

Source

405

[14]

695

*KT881396

239

* AF485804

518

* KX447585

196

*FJ546461

1451

*KJ623587

*KX230478

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S. epidermidis

Sh-R

CCTCCTGTCGTCACCCAATC

16S

Sc-F

AAGTCGAGCGAACTGACGAG

rRNA

Sc-R

TCGTTTACGGCGTGGACTAC

rdr

SERF

AAGAGCGTGGAGAAAAGTATCAAG

SERR

TCGATACCATCAAAAAGTTGG

768

*AJ343945

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[15]

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S. chromogenes

rRNA

* Designed for the current study using source DNA sequences retrieved from NCBI

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2.4. Optimization of monoplex PCR assays

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For optimization of monoplex PCR assays, varying concentrations of magnesium (1.5, 2.0 and

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2.5 mM), Taq DNA polymerase (1.0, 1.5, 2.0, and 2.5 U per reaction), and primers (0.5, 1.0, and

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1.5 µM) were used in 25 µL reaction volume. Every PCR reaction mixture contained 200 µM

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dNTPs and 100-200 ng of template DNA and was thermally cycled once at 94oC for 5 min,

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followed by 30 cycles at 94oC for 30 s; 58oC for 30 s, 72oC for 1 min and finally once at 72oC for

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10 min. The amplified products were electrophoresed through 1.2% agarose gel containing

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ethidium bromide and analyzed under UV light.

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2.5. Optimization of multiplex PCR Assay

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To optimize the multiplex PCR assay, different combinations of individual PCRs with varying

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concentrations of primers and template DNA were used. The final protocol included all the nine

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sets of primer pairs, every set targeting a unique mastitic pathogen. Different concentrations of

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primers were used to obtain specific results. Every multiplex PCR reaction mixtures contained

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1× HotStartTaq Master Mix (Qiagen, Mississauga, ON), 5 µL of primer mix, 50-200 ng of

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template DNA in 50 µL reaction volume The PCR conditions included an initial activation step

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at 95°C for 5 min, followed by 10 cycles of amplification (95°C for 30 s, 62-53°C (1°C decrease

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in every cycle) for 30 s, and 72°C for 1 min), 25 cycles of further amplification (95°C for 30 s,

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53°C for 30 s, and 72°C for 1 min) and a final extension step at 72°C for 10 min.

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2.6. Analytic sensitivity of multiplex PCR assay

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DNA extracted from bacterial cultures of target species was quantified using Nanodrop (Thermo

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Scientific 2000 spectrophotometer) and serially diluted in the range of 100 ng to 0.01 pg using

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nuclease free water. PCR reactions were performed for all these DNA concentrations to

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determine the sensitivity of primers. The limit of detection (LOD) of multiplex PCR assay was

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tested on artificial blends of serially diluted DNA samples.

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To further assess the LOD of the developed assay, pasteurized milk was spiked with all the target

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bacterial species individually as well as in random combinations up to three species per reaction.

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Serial dilutions of spiked milk were made in the range of 106 CFU/ml to 100 CFU/ml. DNA was

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isolated from these dilutions of spiked milk and amplified under optimized conditions. Un-

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inoculated pasteurized milk served as a negative control.

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2.7. Sample collection

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A total of 223 milk samples were collected aseptically from dairy cows and buffalos suspected of

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being affected by mastitis. All these milk samples were stored at -20oC until further analysis.

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2.8. Surf field mastitis test and California mastitis test

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Three percent Surf solution was prepared by dissolving 3 g of commonly used detergent powder

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(Surf Excel, UnileverTM, Pakistan) in 100 ml final volume. Milk samples and Surf solution were

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mixed in equal quantities in Petri dishes. The formation of gel indicated the status of mastitis

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[16]. For California mastitis test, a four-well plastic paddle was used. Equal volumes of the test

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reagent and milk samples were added in each well and gently agitated. The reaction was scored

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on a scale of 0 (mixture remains unchanged) to 3 (almost-solid gel forms), with a score of 2 or 3

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being considered a positive result [17].

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2.9. Bacterial culture from milk samples

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Bacterial culturing was performed according to standards listed by Hogan et al. [18]. Ten

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microliters of each milk sample was cultured on blood agar, MacConkey’s agar and nutrient

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agar. The inoculated plates were incubated at 37oC for 24-48 hrs. The bacterial isolates were

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identified on the basis of their cultural and morphological characteristics, gram staining and

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hemolytic patterns. Single defined colonies were picked and cultured in nutrient broth at 37oC

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overnight in a shaking incubator. DNA was isolated from the bacterial cultures through an

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organic DNA extraction method [19].

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2.10. Milk bacterial DNA extraction and quantification

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Bacterial DNA was extracted directly from milk following the method described by Cremonesi

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et al. [20], with slight modifications. Instead of 500 µL, 3 ml of milk was used and the amount of

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lysis buffer and binding solutions was adjusted accordingly. DNA quantification was done by

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optical density measurements using Nanodrop (Thermo Scientific 2000 spectrophotometer).

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Working DNA dilutions were prepared and stored at -20oC until further use.

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2.11. PCR amplification and DNA sequencing

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For species identification of mastitic milk cultures, a set of universal primers (27F & 1492R)

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targeting the 16S rRNA gene was used [21]. PCR was performed on DNA isolated from bacterial

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cultures as well as mastitic milk samples. Detection of PCR products was carried out by agarose

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gel electrophoresis. PCR products were purified and sequenced through Sanger dideoxy

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sequencing (ABI Genetic Analyzer, 3130xl, Life Technologies) and results were analyzed

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through various bioinformatics tools including Chromas Lite 2.1 software (Technelysium Pty

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Ltd, Australia).

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2.12. Pathogen detection in milk samples by multiplex PCR assay

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The target mastitic pathogens were detected from randomly collected milk samples using the

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developed multiplex PCR assay. The obtained results were compared with those of culture

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method and 16S rRNA sequence analysis.

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2.13. Statistical analysis

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The specificities and sensitivities of the developed multiplex PCR assay, bacterial culture and

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16S rRNA sequencing were estimated for every target species by applying LCA [11]. The LCA

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was carried out in OpenBUGS version 3.2.2 rev 1063 [22] and R version 3.0.2 [23]. In LCA, all

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the parameters need to have their prior distributions specified. However, in the current study, no

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prior information was assumed so uninformative priors in the form of uniform distribution, i.e.

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Beta (1,1) distribution, were specified (three Se, three Sp and prevalence) to avoid any bias. The

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Bayesian method uses a Markov Chain Monte Carlo (MCMC) sampling algorithm to

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numerically approximate the posterior distribution. To allow convergence, the first 10,000

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samples were discarded as burn-in; the following 50,000 iterations were used for posterior

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inference. The MCMC chain, after initial burn in, was assessed by visual inspection of time-

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series plots of variables [24]. Posterior inference was estimated in the form of mean, with

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associated standard error (standard deviation of the MCMC iterations). The possible

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combinations of the target species for the three tests were +++, ++-, +-+, +--, -++, -+-, --+, ---,

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depending on if diagnostic tests were positive or negative for 16S rRNA sequence analysis,

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bacterial culture, and multiplex PCR assay, respectively.

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3. Results

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3.1. Optimization of monoplex PCR assays

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All the reference strains were specifically amplified by monoplex PCRs. There was no prominent

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difference in optimized conditions of monoplex PCR assays. The final reaction mixture for every

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monoplex PCR contained 2U of Taq DNA polymerase, 1.5 mM MgCl2 and 1µM primer

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concentration. PCR product sizes obtained for S. epidermidis, E. coli, S. uberis, M. bovis, S.

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agalactiae, S. aureus, S. dysagalactiae, S. chromogenes and S. haemolyticus were 130, 196, 239,

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343, 405, 518, 695, 768 and 1451 bps, respectively, as shown in Figure 1.

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1.5kb

600bps

100bps

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1: S. epidermidis (130 bp), Lane 2: E. coli (196 bp), Lane 3: S. uberis (239 bp), Lane 4: M. bovis (343

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bp), Lane 5: S. agalactiae (405 bp), Lane 6: S. aureus (518 bp), Lane 7: S. dysagalactiae (695 bp), Lane

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8: S. chromogenes (768 bp)

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3.2. Optimization of multiplex PCR assay

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Different combinations of monoplex PCR assays were tested and finally all the nine target

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pathogens were detected in a single reaction. When the primers were added in equal

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concentrations, few of the target species were not detected. The final primer mix included primer

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pairs in the following concentrations: Mb (0.45 µM), Sg (0.30 µM), Sd (0.50 µM), Su (0.40

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µM), Sa (0.35 µM), Ec (0.55 µM), Sh (0.30 µM), Sc (0.45 µM) and SERF (0.25 µM). All the

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primers targeting the 16S rRNA, PhoA, Cpn60 and rdr genes were species-specific and they

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efficiently detected the target pathogens as shown in Figure 2.

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100bps

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Figure 2. Multiplex PCR assay for mastitic bacterial pathogens. Lane L: 100 bp DNA ladder, Lane 1: S.

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epidermidis (130 bp), & S. uberis (239 bp), Lane 2: E. coli (196 bp) & M. bovis (343 bp), Lane 3: S.

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uberis (239 bp) & S. agalactiae (405 bp), Lane 4: E. coli (196 bp) & S. aureus (518 bp), Lane 5: M. bovis

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(343 bp), S. dysagalactiae (695 bp) & S. chromogenes (768 bp), Lane 6: S. uberis (239 bp), S. aureus

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(518 bp) & S. chromogenes (768 bp), Lane 7: E. coli (196 bp), S. chromogenes (235 bp), S. agalactiae

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(405 bp), S. dysagalactiae (695 bp) & S. haemolyticus (1451 bp), Lane 8: E. coli (196bp), M. bovis (343

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bp), S. aureus (518 bp), S. chromogenes (768 bp) & S. haemolyticus (1451 bp), Lane 9: all the nine

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pathogens.

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3.3. Analytic sensitivity of multiplex PCR assay

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The sensitivity of monoplex PCR assay for each target bacteria was higher as compared to

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duplex or triplex PCR assays. S. aureus, S. uberis, S. chromogenes and S. dysgalactiae were

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detected at DNA concentration as low as 0.1 pg, S. agalactiae, S. haemolyticus and M. bovis

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were detected at 1 pg or higher DNA concentration, while E. coli and S. epidermidis were

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detected at 10 pg of DNA concentration. Multiplex PCR assay was comparatively less sensitive

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than monoplex PCR assays. The LOD of multiplex PCR assay was in the range of 1.0 to 50 pg

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DNA for all possible random combinations of the target species. DNA samples isolated from

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milk samples spiked with different combinations of the target species were amplified with

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different sensitivities. The LOD for S. aureus, S. dysgalactiae, S. agalactiae & S. chromogenes

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was 101 CFU/ml; E. coli, S. uberis & M. bovis were detected at 102 CFU/ml, while S.

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epidermidis & S. haemolyticus were detected at 103 CFU/ml. Sensitivity for random

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combinations of two or three bacterial species was in the range of 101 to 104 CFU/ml as shown in

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Table 2.

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E. coli

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Table 2. Analytic sensitivity of multiplex PCR assay Bacterial species

Sensitivity pg/ml (serial dilutions of DNA from pure cultures) Individual PCR Multiplex PCR 10 10

S. aureus

0.1

S. agalactiae

1

Sensitivity CFU/ml (serial dilutions of inoculated milk samples) Individual PCR Multiplex PCR 102 102

1

101

102

1

101

101

1

101

102

10

103

104

0.1

S. haemolyticus

1

S. chromogenes

0.1

10

101

102

S. epidermidis

10

50

103

104

M. bovis

1

10

102

104

S. uberis

0.1

10

102

103

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S. dysgalactiae

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3.4. Pathogen detection by 16S rRNA sequencing

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The detection of bacterial pathogens based on 16S rRNA sequencing was considered as gold

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standard to compare the results of the developed multiplex PCR assay with those of bacterial

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culture. The results of California mastitis test showed that out of 223 milk samples, 200 were

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positive for mastitis. Among these positive samples, 158 were found to have one or two target

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pathogens, totaling to 276 different bacterial strains out of which 198 were identified as

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Staphylococci, Streptococci, and E. coli. Six milk samples showed no growth on any growth

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media. M. bovis was not recovered from any of the milk samples. On the basis of 16S rRNA

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sequencing, 58 bacterial isolates were identified as S. aureus, 37 as E. coli, 29 as S. uberis, 27 as

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S. dysagalactiae, 23 as S. agalactiae, 9 as S. haemolyticus, 8 as S. chromogenes, 7 as S.

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epidermidis, 14 as S. simulans, 20 as S. pyogenes, 24 as Bacillus sp. and 15 as Corynebacterium

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sp. Bacterial species other than these common mastitic pathogens were also detected from the

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milk samples, including Bacillus jeotgali, Staphylococcus saprophyticus, Corynebacterium

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confusum, Brevibacillus formosus and Escherichia fergusonii.

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3.5. Pathogen detection in milk samples by multiplex PCR assay

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After being validated on DNA isolated from milk cultures, multiplex PCR assay was tested to

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detect the target pathogens by using DNA extracted directly from milk. The assay achieved

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efficient identification of the target bacteria, as shown in Figure 3. S. aureus, E. coli, S. uberis, S.

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dysagalactiae, S. agalactiae, S. haemolyticus, S. chromogenes and S. epidermidis were detected

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for 57, 37, 29, 27, 23, 9, 8 and 7 times, respectively. None of the milk samples under study was

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positive for M. bovis. According to the multiplex PCR assay, 54.5% of the total milk samples

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were positive for a single target species, 24.5% were positive for more than one target species,

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whereas 21% were negative for all the target bacterial species. S. aureus & S. uberis, E. coli & S.

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uberis, E. coli & S. dysagalactiae, S. epidermidis & S. agalactiae, S. epidermidis & S.

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dysagalactiae, and S. aureus & E. coli were detected in the form of combined bovine

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intramammary infection.

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1

2

3

4

5

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7

8

9

10 11 12

13 14

15

16

17

2.0kb

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500bp

100bp

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Figure 3. Identification of bacterial pathogens from milk by multiplex PCR assay. Lane L: 100 bp DNA

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ladder, Lanes 1 & 2: E. coli (196 bp), Lanes 3 & 4: S. epidermidis (130 bp), Lanes 5 & 6: S. agalactiae

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(405 bp), Lanes 7 & 8: S. dysagalactiae (695 bp), Lanes 9 & 10: S. epidermidis (130 bp), Lanes 11 & 12:

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E. coli (196 bp), Lanes 13 & 14: S. aureus (518 bp), Lane 15: S. agalactiae (405 bp) & S. epidermidis

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(130 bp), Lane 16: S. dysagalactiae (695 bp) & S. epidermidis (130 bp), Lane 17: E. coli (196 bp) & S.

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aureus (518 bp)

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3.6. Sensitivity and specificity estimation by LCA

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Table 3 shows the frequency distribution of all the combinations of the test results for each target

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species. The relatively high frequencies for entries ‘+ + +’ and ‘− − −’ indicate an overall high

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agreement between the test results of the three diagnostic assays used.

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Test combinations

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Table 3. The test results for 16S rRNA sequence analysis, bacterial culture, and multiplex PCR assay +−+

+−−

−++

−+−

−−+

−−−

0

12

0

1

3

0

139

31

0

6

1

0

2

0

160

S. uberis

23

0

6

0

0

1

0

170

S. dysagalactiae

19

0

8

0

0

4

0

169

S. agalactiae

19

0

4

0

0

2

0

175

S. haemolyticus

6

0

3

1

0

0

0

190

S. chromogenes

6

0

2

1

0

0

0

191

S. epidermidis

5

0

2

1

0

0

0

192

Species

E. coli

++−

45

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S. aureus

+++

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The LCA sensitivity and specificity estimates for 16S rRNA sequence analysis, bacterial culture

278

method and multiplex PCR assay are given in Table 4. The results showed that sensitivity and

279

specificity estimates were highest for 16S rRNA sequence analysis followed by multiplex PCR

280

assay; bacterial culture method showed lower values for both. As the sensitivity and specificity

281

estimates were closer to 1.0 in the case of 16S rRNA sequence analysis, it can be assumed as a

282

gold standard.

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16S rRNA

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Table 4. Mean values of LCA specificity/sensitivity estimates for targeted mastitic pathogens Bacterial Culture SE

Mean

SE

Mean

SE

S. aureus

0.992/0.966

0.007/0.023

0.972/0.784

0.014/0.052

0.992/0.982

0.007/0.017

E. coli

0.989/0.974

0.008/0.026

0.982/0.816

0.010/0.062

0.993/0.969

0.006/0.029

S. uberis

0.994/0.967

0.006/0.032

0.988/0.774

0.008/0.073

0.994/0.967

0.006/0.032

S. dysagalactiae

0.994/0.964

0.006/0.034

0.971/0.690

0.013/0.084

0.994/0.964

0.006/0.035

S. agalactiae

0.994/0.957

0.006/0.040

0.983/0.798

0.010/0.079

0.994/0.957

0.006/0.040

S. haemolyticus

0.990/0.905

0.007/0.086

0.994/0.622

0.005/0.141

0.994/0.890

0.005/0.096

S. chromogenes

0.990/0.898

0.007/0.091

0.994/0.685

0.005/0.142

0.994/0.880

0.005/0.103

S. epidermidis

0.990/0.886

0.005/0.152

0.994/0.862

0.005/0.115

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Bacterial Sample

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Mean

Multiplex PCR Assay

0.007/0.101

0.994/0.648

4. Discussion

285

The current study developed a novel multiplex PCR assay for robust and accurate detection of

286

nine bacterial pathogens frequently associated with bovine mastitis. Accurate diagnosis is an

287

important step between cause and cure of the disease. The development of an economical, simple

288

and rapid diagnostic tool has always been fundamental for the management of udder health [25].

289

Sequence analyses of conserved “housekeeping” genes such as the bacterial 16S rRNA gene are

290

increasingly being used to identify bacterial species in clinical practice and scientific

291

investigations [26]. In the case of 16S rRNA analysis, species identification is easiest when most

292

or the entire gene can be sequenced. 16S rRNA gene sequencing allows robust, highly

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reproducible and accurate identification of bacteria and is, therefore, favored over phenotypic

294

testing. In addition, 16S rRNA sequencing allows discovery of novel, clinically relevant bacteria

295

and the test results are objective [27]. DNA sequencing is, however, impractical in medical

296

diagnostics where assay time and speed are often of essence [28]. Also DNA sequencing

297

becomes expensive when individual tests for every target species are performed for mastitis

298

cases involving more than single pathogens. Based on this rationale, our research focused on the

299

development of multiplex PCR assay capable of detecting multiple pathogens in a single

300

reaction.

301

Primers were designed from the 16S rRNA gene for M. bovis, S. agalactiae, S. dysagalactiae, S.

302

chromogenes, S. haemolyticus and S. aureus, from the PhoA gene for E. coli, the Cpn60 gene for

303

S. uberis and the rdr gene for S. epidermidis. Selection was based on the specificity, Tm and

304

difference in amplicon size. All the primers were tested with reference strains for the validation

305

of the assay. E. coli shows great homology in sequence with other species of Enterobacteriaceae,

306

so multiple targets are required for its identification [29]. Because it was not possible to target

307

multiple genes for E. coli in our multiplex PCR assay, we have chosen PhoA gene for designing

308

primers which specifically detect E. coli.

309

When the multiplex PCR assay was tested to detect pathogens from mastitic milk, it proved to be

310

very efficient and accurate. The assay detected all the bacterial species isolated by culture

311

method, and identified the pathogens in the culture-negative samples too. In subclinical mastitis,

312

samples with no bacterial growth are generally very common. Microorganisms do not exist as

313

pure culture but appear in the form of complex communities. The existing methods in clinical

314

microbiology lack the ability to rapidly catalog and comprehensively classify the diversity of

315

organisms present in case specimens [30]. Individual strains may surpass others when co-

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cultured, and overwhelming numbers of species may be present, prohibiting a comprehensive

317

workup. The conventional method for bacterial identification is based on bacterial culturing,

318

followed by examination of phenotypic, biochemical, and enzymatic characteristics of bacteria.

319

However, the microbial culture of milk samples presents many limitations [31]. Most bacterial

320

species were detected by the bacterial culture method, but where not detected, was probably due

321

to either absence of viable bacterial cells or due to the fact that bacterial colonies are selected on

322

the basis of morphological features, and hence there is a chance of missing a species which has a

323

similar phenotype. Microbial culturing is considered as a gold standard but it has its own

324

limitations, rendering it imperfect as a true reference test.

325

The analytic sensitivity of multiplex PCR assay reported by Lee et al. [32] ranged from 105

326

CFU/ml to 102 CFU/ml which is comparable to previous studies. However, it was slightly lower

327

as compared to another multiplex PCR assay whose detection limit was in the range of 102

328

CFU/ml to 100 CFU/ml [15]. The analytic sensitivity of our multiplex PCR assay was up to 50 pg

329

for DNA isolated from pure cultures and 104 CFU/ml for spiked milk samples which is

330

comparable to other developed assays. According to Reher et al. [33], if more than two different

331

bacterial species are detected in a single mastitic milk sample it is marked as contaminated, with

332

an exception of S. aureus and S. agalactiae. In this regard, we combined a maximum of two or

333

three target bacterial strains randomly to check the sensitivity of multiplex PCR assay. In a

334

recently reported study, 33.5% of quarters were infection free; however, single pathogens were

335

detected in 43.5% of mastitis positive quarters, two bacterial species were identified in 19.2% of

336

infection and very small percentage of infections 3.8% were due to three pathogens [34].

337

Somewhat similar results had also been reported in another study [35]. Detection limit of

338

multiplex PCR assay is usually low as compared to monoplex PCR. In a study designed for

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detection of S. aureus and Yersinia enterocolitica, the detection limit of multiplex PCR assay

340

applied on DNA isolated from bacterial culture was 100 CFU/ml. However, it decreased to 103

341

CFU/ml and 104 CFU/ml when DNA obtained from spiked milk samples was subjected to

342

monoplex and multiplex PCR assays, respectively [36].

343

The sensitivity and specificity of a test are usually determined by comparison with a reference

344

test which is supposed to determine the true disease state of the animals unambiguously. But this

345

is only possible in the presence of perfect reference test [37]. The true disease state, however, is

346

rarely known in practice, because perfect test results may be difficult or impossible to obtain.

347

The sensitivity and specificity were estimated by applying LCA model for evaluation of the

348

developed multiplex PCR assay. The results showed that multiplex PCR assay had higher

349

sensitivity and specificity as compared to bacterial culture for all the target species. It has been

350

elaborated that a PCR assay can efficiently detect DNA from both viable and nonviable bacteria

351

while bacterial culture is only capable of detecting viable bacteria. In those cases where the

352

infection is cured, bacterial DNA can still be detected in the udder and false positive PCR assay

353

results can be obtained [8].

354

The sensitivity and specificity of a real-time PCR assay for the detection of bovine mastitic

355

pathogens was found to be 100% for almost all the target species [38]. In another study, a rapid

356

PCR test was developed for the identification of S. agalactiae in milk samples collected on filter

357

paper disks. The diagnostic sensitivity of the test was 96.15% and specificity was 98.60% [39].

358

Paradis et al. [40] used an LCA model for estimation of sensitivity and specificity of multiplex

359

real-time PCR assay for the detection of S. aureus, S. uberis, E. coli, and S. agalactiae.

360

Sensitivity was found to be in the range of 66% to 96% and specificity was ≥ 99% for all the

361

target species. In the present study, similar levels of test performance for multiplex PCR assay

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have been achieved; however, our assay is capable of detecting nine instead of four bacterial

363

species.

364

5. Conclusion

365

To the best of our knowledge, the developed multiplex PCR assay is the first to achieve a low-

366

cost, high-throughput capacity, and fast turnaround time for simultaneous detection of nine

367

bacterial mastitic pathogens and appears to be sufficiently specific and sensitive for disease

368

diagnosis and the target species differentiation. The specificities and sensitivities estimated by

369

LCA clearly indicate that the assay has a similar performance level to the 16S rRNA sequence

370

analysis; however, it is far more rapid to perform. This novel molecular assay could be helpful

371

for correct and timely identification of bovine mastitic pathogens, which is crucial for the control

372

and treatment of the disease

373

6. Declarations

374

6.1. Ethics approval and consent to participate

375

Not applicable

376

6.2. Consent for publication

377

Not applicable

378

6.3. Availability of data and material

379

Not applicable

380

6.4. Competing interests

381

Authors declare no competing interests.

382

6.5. Funding

383

This work was supported by the Higher Education Commission of Pakistan under the

384

International Research Support Initiative Program (IRSIP).

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6.6. Authors' contributions

386

MI, TY, MT and AA conceived and designed the study, AA carried out experiments and wrote

387

the manuscript; MI and WS supervised the work; PT carried out statistical analysis; MI, WS and

388

PT did proof reading of the manuscript; all authors read and approved the final manuscript.

389

6.7. Acknowledgements

390

Authors are thankful to Shumaila Arif, School of Animal and Veterinary Sciences, Charles Sturt

391

University, Wagga Wagga, Australia for providing assistance in statistical work and proof

392

reading of paper. Special thanks to Quality Milk Production Services (QMP) Cornell University,

393

Ithaca, NY, USA for providing reference strains for analysis.

394

7. References

395

[1]

M AN U

SC

RI PT

385

R. Hussain, M.T. Javed, A. Khan, Changes in some biochemical parameters and somatic cell counts in the milk of buffalo and cattle suffering from mastitis, Pak. Vet. J. 32 (2012)

397

418–421.

398

[2]

TE D

396

Y. Kuang, K. Tani, A.J. Synnott, K. Ohshima, H. Higuchi, H. Nagahata, Y. Tanji, Characterization of bacterial population of raw milk from bovine mastitis by culture-

400

independent PCR–DGGE method, Biochem. Eng. J. 45 (2009) 76–81. [3]

O.M. Radostitis, C.C. Gay, D.C. Blood, K.W. Hinchcliff, Veterinary medicine 9th ed, WB

[4]

404

AC C

EP

399

405

involved in bovine mastitis, Vet. Microbiol. 106 (2005) 61–71.

401

Sauders. London. (2000).

402 403

406 407

J. dos Santos Nascimento, P.C. Fagundes, M.A.V. de Paiva Brito, K.R.N. Dos Santos, M. do C. de Freire Bastos, Production of bacteriocins by coagulase-negative staphylococci

[5]

H. Hiitiö, R. Riva, T. Autio, T. Pohjanvirta, J. Holopainen, S. Pyörälä, S. Pelkonen, Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared 20

ACCEPTED MANUSCRIPT

408

with in-depth conventional culture, J. Dairy Res. 82 (2015) 200–208.

409

doi:10.1017/S0022029915000084. [6]

C. Viguier, S. Arora, N. Gilmartin, K. Welbeck, R. O’Kennedy, R. O’Kennedy, Mastitis

RI PT

410 411

detection: current trends and future perspectives, Trends Biotechnol. 27 (2009) 486–493.

412

doi:10.1016/j.tibtech.2009.05.004. [7]

R. Biek, A. O’Hare, D. Wright, T. Mallon, C. McCormick, R.J. Orton, S. McDowell, H.

SC

413

Trewby, R.A. Skuce, R.R. Kao, Whole genome sequencing reveals local transmission

415

patterns of Mycobacterium bovis in sympatric cattle and badger populations, PLoS

416

Pathog. 8 (2012) e1003008.

417

[8]

M AN U

414

M.T. Koskinen, G.J. Wellenberg, O.C. Sampimon, J. Holopainen, a Rothkamp, L. Salmikivi, W. a van Haeringen, T.J.G.M. Lam, S. Pyörälä, Field comparison of real-time

419

polymerase chain reaction and bacterial culture for identification of bovine mastitis

420

bacteria., J. Dairy Sci. 93 (2010) 5707–15. doi:10.3168/jds.2010-3167.

421

[9]

TE D

418

G. Matope, J.B. Muma, N. Toft, E. Gori, A. Lund, K. Nielsen, E. Skjerve, Evaluation of sensitivity and specificity of RBT, c-ELISA and fluorescence polarisation assay for

423

diagnosis of brucellosis in cattle using latent class analysis, Vet. Immunol. Immunopathol.

424

141 (2011) 58–63. doi:10.1016/j.vetimm.2011.02.005.

[10]

Vet. Med. 77 (2006) 96–108.

427

429

C.J. Sanford, G.P. Keefe, J. Sanchez, R.T. Dingwell, H.W. Barkema, K.E. Leslie, I.R. Dohoo, Test characteristics from latent-class models of the California Mastitis Test, Prev.

426

428

AC C

425

EP

422

[11]

S.L. Hui, S.D. Walter, Estimating the error rates of diagnostic tests, Biometrics. (1980) 167–171.

21

ACCEPTED MANUSCRIPT

[12]

practice and research, Aust. Vet. J. 80 (2002) 758–761.

431 432

[13]

A. Untergasser, I. Cutcutache, T. Koressaar, J. Ye, B.C. Faircloth, M. Remm, S.G. Rozen, Primer3—new capabilities and interfaces, Nucleic Acids Res. 40 (2012) e115–e115.

433 434

I.A. Gardner, The utility of Bayes’ theorem and Bayesian inference in veterinary clinical

RI PT

430

[14]

R. Riffon, K. Sayasith, H. Khalil, P. Dubreuil, M. Drolet, J. Lagace, Development of a Rapid and Sensitive Test for Identi cation of Major Pathogens in Bovine Mastitis by PCR

436

´, Society. 39 (2001) 2584–2589. doi:10.1128/JCM.39.7.2584. [15]

B.R. Shome, S. Das Mitra, M. Bhuvana, N. Krithiga, D. Velu, R. Shome, S. Isloor, S.B.

M AN U

437

SC

435

438

Barbuddhe, H. Rahman, Multiplex PCR assay for species identification of bovine mastitis

439

pathogens, J. Appl. Microbiol. 111 (2011) 1349–1356. doi:10.1111/j.1365-

440

2672.2011.05169.x. [16]

G. Muhammad, M. Athar, A. Shakoor, M.Z. Khan, F. Rehman, M.T. Ahmad, Surf Field

TE D

441 442

Mastitis Test: An inexpensive new tool for evaluation of wholesomeness of fresh milk,

443

Pakistan J. Food Sci. 5 (1995) 91–93.

Test, J. Dairy Res. 75 (2008) 385–391.

445 446

[18]

(1999) 6–10.

448

[19]

D.W. Sambrook JaR, Molecular Cloning: A Laboratory Manual. 3re ed Cold Spring Harbor Laboratory Press, (2001).

450 451

J.S. Hogan, R.N. Gonzalez, R.J. Harmon, S.C. Nickerson, S.P. Oliver, J.W. Pankey, K.L. Smith, Laboratory handbook on bovine mastitis, Natl. Mastit. Counc. Madison, WI.

447

449

C.J.R. Verbeek, S.S. Xia, D. Whyte, Rheology of the gel formed in the California Mastitis

EP

[17]

AC C

444

[20]

P. Cremonesi, B. Castiglioni, G. Malferrari, I. Biunno, C. Vimercati, P. Moroni, S. 22

ACCEPTED MANUSCRIPT

Morandi, M. Luzzana, Technical note: Improved method for rapid DNA extraction of

453

mastitis pathogens directly from milk., J. Dairy Sci. 89 (2006) 163–9.

454

doi:10.3168/jds.S0022-0302(06)72080-X.

455

[21]

RI PT

452

H. Jiang, H. Dong, G. Zhang, B. Yu, L.R. Chapman, M.W. Fields, Microbial diversity in water and sediment of Lake Chaka, an athalassohaline lake in northwestern China, Appl.

457

Environ. Microbiol. 72 (2006) 3832–3845. [22]

D.J. Lunn, A. Thomas, N. Best, D. Spiegelhalter, WinBUGS-a Bayesian modelling framework: concepts, structure, and extensibility, Stat. Comput. 10 (2000) 325–337.

459

M AN U

458

SC

456

460

[23]

R Core Team, R: A language and environment for statistical computing, (2013).

461

[24]

N. Toft, J. Åkerstedt, J. Tharaldsen, P. Hopp, Evaluation of three serological tests for diagnosis of Maedi-Visna virus infection using latent class analysis, Vet. Microbiol. 120

463

(2007) 77–86. doi:10.1016/j.vetmic.2006.10.025.

464

[25]

TE D

462

Y.S. Mustafa, F.N. Awan, T. Zaman, Prevalence and antibacterial susceptibility in mastitis in buffalo and Cow in district Lahore-Pakistan, Buffalo Bull. 32 (2013) 307–314.

466

http://ibic.lib.ku.ac.th/e-bulletin/IBBU201304010.pdf. [26]

C. a Petti, C.R. Polage, P. Schreckenberger, The role of 16S rRNA gene sequencing in

[27]

AC C

467

identification of microorganisms mis-identifed by conventional methods, Society. 43

468

(2005) 6123–6125. doi:10.1128/JCM.43.12.6123.

469 470

EP

465

P.C.Y. Woo, S.K.P. Lau, J.L.L. Teng, H. Tse, K.-Y. Yuen, Then and now: use of 16S

471

rDNA gene sequencing for bacterial identification and discovery of novel bacteria in

472

clinical microbiology laboratories, Clin. Microbiol. Infect. 14 (2008) 908–934.

473

doi:10.1111/j.1469-0691.2008.02070.x. 23

ACCEPTED MANUSCRIPT

474

[28]

S. Chakravorty, D. Helb, M. Burday, N. Connell, A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria, J Microbiol Methods. 69

476

(2007) 330–339. doi:10.1016/j.mimet.2007.02.005.A.

477

[29]

RI PT

475

K. Horakova, H. Mlejnkova, P. Mlejnek, Specific detection of Escherichia coli isolated from water samples using polymerase chain reaction targeting four genes: cytochrome bd

479

complex, lactose permease, β‐d‐glucuronidase, and β‐d‐galactosidase, J. Appl. Microbiol.

480

105 (2008) 970–976. [30]

R. Schlaberg, K.E. Simmon, M.A. Fisher, A systematic approach for discovering novel,

M AN U

481

SC

478

482

clinically relevant bacteria, Emerg. Infect. Dis. 18 (2012) 422–430.

483

doi:10.3201/eid1803.111481.

484

[31]

S. Taponen, L. Salmikivi, H. Simojoki, M.T.T. Koskinen, S. Pyörälä, Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine

486

clinical mastitis with no growth in conventional culturing., J. Dairy Sci. 92 (2009) 2610–7.

487

doi:10.3168/jds.2008-1729. [32]

K.-H. Lee, J.-W. Lee, S.-W. Wang, L.-Y. Liu, M.-F. Lee, S.-T. Chuang, Y.-M. Shy, C.-L.

EP

488

TE D

485

Chang, M.-C. Wu, C.-H. Chi, Development of a novel biochip for rapid multiplex

490

detection of seven mastitis-causing pathogens in bovine milk samples, J. Vet. Diagnostic Investig. 20 (2008) 463–471. doi:10.1177/104063870802000408.

491 492

AC C

489

[33]

K.K. Reyher, S. Dufour, H.W. Barkema, L. Des Côteaux, T.J. Devries, I.R. Dohoo, G.P.

493

Keefe, J.-P. Roy, D.T. Scholl, The National Cohort of Dairy Farms—A data collection

494

platform for mastitis research in Canada, J. Dairy Sci. 94 (2011) 1616–1626.

495

[34]

M. Goli, H. Ezzatpanah, M. Ghavami, M. Chamani, M. Aminafshar, M. Toghiani, S.

24

ACCEPTED MANUSCRIPT

Eghbalsaied, The effect of multiplex-PCR-assessed major pathogens causing subclinical

497

mastitis on somatic cell profiles, Trop. Anim. Health Prod. 44 (2012) 1673–1680.

498

doi:10.1007/s11250-012-0123-3.

499

[35]

D. Rysanek, V. Babak, M. Zouharova, Bulk tank milk somatic cell count and sources of raw milk contamination with mastitis pathogens, Vet. Med. (Praha). 52 (2007) 223–230.

500

[36]

a Ramesh, B.P. Padmapriya, a Chrashekar, M.C. Varadaraj, Application of a convenient

SC

501

RI PT

496

DNA extraction method and multiplex PCR for the direct detection of Staphylococcus

503

aureus and Yersinia enterocolitica in milk samples., Mol. Cell. Probes. 16 (2002) 307–

504

314. doi:S0890850802904287 [pii].

505

[37]

H.C. Kraemer, Evaluating medical tests: Objective and quantitative guidelines, Sage publications Newbury Park, CA:, 1992.

506

[38]

M.T. Koskinen, J. Holopainen, S. Pyörälä, P. Bredbacka, a Pitkälä, H.W. Barkema, R.

TE D

507

M AN U

502

Bexiga, J. Roberson, L. Sølverød, R. Piccinini, D. Kelton, H. Lehmusto, S. Niskala, L.

509

Salmikivi, Analytical specificity and sensitivity of a real-time polymerase chain reaction

510

assay for identification of bovine mastitis pathogens., J. Dairy Sci. 92 (2009) 952–9.

511

doi:10.3168/jds.2008-1549. [39]

Streptococcus agalactiae in milk samples collected on filter paper disks, Asian-

513

Australasian J. Anim. Sci. 21 (2008) 124–130.

514 515

J. Wu, Y. Liu, S. Hu, J. Zhou, Development of a rapid PCR test for identification of

AC C

512

EP

508

[40]

M.-È. Paradis, D. Haine, B. Gillespie, S.P. Oliver, S. Messier, J. Comeau, D.T. Scholl,

516

Bayesian estimation of the diagnostic accuracy of a multiplex real-time PCR assay and

517

bacteriological culture for 4 common bovine intramammary pathogens., J. Dairy Sci. 95

25

ACCEPTED MANUSCRIPT

518

(2012) 6436–48. doi:10.3168/jds.2012-5328.

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Highlights •

A single reaction based multiplex PCR assay was developed and evaluated for rapid identification of nine mastitis causing bacterial pathogens. The analytic sensitivity of the assay was up to 50 pg for DNA isolated from pure cultures and 104 CFU/ml for spiked milk samples.

The developed assay has sensitivity ≥ 88% and specificity ≥ 98% targeted pathogens as

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estimated by latent class analysis.

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•