Simultaneous detection of six human diarrheal pathogens by using DNA microarray combined with tyramide signal amplification

Journal of Microbiological Methods 75 (2008) 365–368

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Simultaneous detection of six human diarrheal pathogens by using DNA microarray combined with tyramide signal amplification Dazhi Jin 1, Hongjuan Qi, Suhong Chen, Ting Zeng, Qiqi Liu, Shengqi Wang ⁎ Beijing Institute of Radiation Medicine, No. 27 Taiping road, Beijing, China

A R T I C L E

I N F O

Article history: Received 21 December 2007 Received in revised form 18 June 2008 Accepted 20 June 2008 Available online 2 July 2008 Keywords: Human diarrheal pathogens Detection DNA microarray Tyramide signal amplification

A B S T R A C T Multiplex PCR and DNA microarray were combined with tyramide signal amplification (TSA) to develop a reliable method suitable for simultaneous detection of six species of human diarrheal pathogens (Yersinia enterocolitica, Shigella spp, Salmonella typhi, Brucella spp, Vibrio cholera and Escherichia coli O157:H7). Meanwhile, our method could distinguish V. cholera serotype O1 from O139, and O157:H7 from O157: nonH7. This assay conferred a specificity of 100% for target pathogens. The limit of detection was 103 °CFU/mL approximately. The results of 98.6% (357/362) clinical specimens and 100% (5/5) mocked double-blind samples were the same to that from conventional assay. Consequently this assay is sensitive and a specific tool suitable for diagnostic detection and surveillance of multiple human pathogens. © 2008 Published by Elsevier B.V.

Diarrhea and enteritis are two major intestinal diseases in clinic, and often lead to clinical complications, such as septicemia, encephalitis and meningitis, etc (Evan, 1998; Fratamico et al., 2005). Recent statistical reports from the Ministry of Health People Public of China have shown that a large portion of incidents related to diarrhea and food poisoning are mainly caused by six species of aforementioned pathogens (Ministry of Health P.R. China, 2006.). Obviously, infection and epidemic of pathogenic bacteria still remains as one of the major threats to human health, and therefore is a severe hygienic problem worldwide so far. Conventional microbiological methods are often limited by the length of time and trivial steps required to complete the assay (Kong et al., 2002). The enzyme-linked immunological protocols (Luk, 1994) and molecular biological assays based on DNA probing (Mooney et al., 1995) or PCR amplification (Gonza'lez et al., 2003; Osorio et al., 2000; Kong et al., 2002; Brasher et al., 1998)had overcome the problems associated with culture-based assays due to their specificity, sensitivity and rapidity. To date, DNA microarray combined with multiplex PCR has been applied widely to detect and identify various genera or species of pathogenic microorganisms (Call et al., 2001; Gonza'lez et al., 2004; Del et al., 2002; Chizhikov et al., 2002; Jin et al., 2006). In this study, we described an assay of the coupled multiplex PCRmicroarray, which employed nine primer sets to detect six species or genus of pathogenic diarrheal microorganisms using a TSA-Cy3 reporting system.

⁎ Corresponding author. Beijing Institute of Radiation Medicine, No. 27 Taiping road, Beijing 100850, China. Tel./fax: +86 10 66932211. E-mail addresses: [email protected] (D. Jin), [email protected] (S. Wang). 1 The author's current institute is Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China. Tel./fax: +86 571 87115285. 0167-7012/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.mimet.2008.06.020

Strains of bacteria used in this study are listed in Table 1. A total of 92 strains of bacteria from 18 genera or species were obtained from National Institute for the Control of Pharmaceutical and Biological Products of China. The local strains of Vibrio cholera O1 (569B), V. cholera Ogawa, V. cholera O139 and Escherichia coli O157: H7, and some intestinal bacteria strains were provided by Zhejiang Provincial Center for Disease Prevention and Control, China. All these selected strains were cultured for 24–36 h according to the conventional method as described (Evan, 1998; Miliotis, 2003). 362 clinical specimens (anus swabs) were provided by Zhejiang provincial Center for Disease Prevention and Control, China. The pathogens that isolated from clinical specimens were identified by the conventional method and the appropriate API test system (bioMerieux SA, Lyon, France) respectively. Meanwhile, 5 mocked double-blind samples containing Yersinia enterocolitica, Brucella and mixed pathogens were prepared. Genomic DNA of bacteria was extracted by centrifugation, lysate buffer (Triton x-100, NP40) as previously described (Jin et al., 2006). All the primer pairs and probes were designed from the specific genes of target pathogens. The corresponding loci chosen were: ail, ipaH, vipR, BCSP, ctxA, LPSgt, rfbE, fliC, and 16S rDNA was used as an internal control. Eight PCR primer sets and eight internal probes were designed using the Primer 5.0 program (PE Biosystems, Foster City, CA). The reverse primers were labeled with biotin group. All the oligonucleotide probes were attached to an amino-modified group at the 3′ end to allow covalent bonding to the aldehyde-coated glass slide. An additional polyethleneglycol spacer was linked to 3′ end of in front of an amino group. Based on the previous study (Jin et al., 2006), we chose a region on 16S rDNA gene as the internal control. DNA microarray was designed to have 10 probes surrounding six columns and five rows included 1 internal control and 1 negative

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Table 1 Reference strains used in this study Species

ATCC accession no.a except particular notes

Salmonella spp

50001, 50004, 50013, 50017, 50019, 50035 50760, 50835, 50041, 50096, 14028 50073, 19430, 6539 14028, 50115 12176, 11511 10719, 19940, 8759 13428, 9068 44752, 43889, 43859, W933, 882364, EDL 933 S14-91, CB569, 5412, 493/89 5905, 5A, 5B 13C07, 13C08 403, 405, TW00186 54003, 54005, 54006, 54007 33090 35967 25401 35897 51081, 51207, 51335, 51424, 35964, 49345, 29029 20502, 20506, 20507, 20511 16025, 16026, 16028 M045, 1837 569B,Ogawa 26001, 26111, 26113,13565,27661 49027, 49101, 49102, 49103 63301,6051,63509 64201 64203 64711,13048 52207, 52211, 52215, 52217, 52302 23456, 23457, 23458, 23459, 11778, 25840 23447, 23448, 23449, 23450, 23365 33560, 7709, 29428, 43429 10501, 35654, 23211, 23213 32219, 32220, 32221, 32223 CMCC (B) 48016 CMCC (B) 45103

Salmonella typhi Salmonella typhimurium Salmonella paratyphi A Salmonella paratyphi B Salmonella paratyphi C Escherichia coli O157:H7 Escherichia coli O157:non-H7 Escherichia coli O55:H7 Escherichia coli O26:H11 Escherichia coli O111:NM Listeria monocytogenes Listeria innocua Listeria seeligeri Listeria grayi Listeria welshimerii Shigella spp Vibrio parahaemolyticus Vibrio cholera Vibrio cholera O139 Vibrio cholera O1 Staphylococcus aureus Proteus spp Bacillus cereus Clostridium botulinum Clostridium perfringens Yersinia enterocolitica Brucella spp Campylobacter jejuni Aeromonas hydrophila Enterococcus faecalis Citrobacter freundii Enterobacter aerogenes

a American Type Culture Collection (ATCC) accession number for positive-control strains.

control. The location spots (complementary sequence of 16S-R) surrounded the array were used to identify the position of each target probe. The layout of DNA microarray was shown in Fig. 1-a. PCR reaction was performed in a volume of 50 μL with 1 × PCR buffer (TaKaRa Biotechology Co. Ltd), 250 μM deoxynucleotide mixture (each of dATP, dUTP, dCTP and dGTP), 20–50 ng of purified genomic

DNA, 2.5 U Taq DNA polymerase (TaKaRa Biotechology Co. Ltd), 3.0 mM MgCl2 and 500 nM each primer. PCR was performed for 35 cycles under the following conditions: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s; and 72 °C for 5 min; 4 °C forever on the DNA thermal system (icycler, Bio-Rad, Hercules, CA, USA). The different ratio of forward to reverse (labeled with biotin) primer of each set was optimized following a value of 1:1, 1:2, 1:3, 1:5, 1:10 and 1:20 respectively. Other components were as above. Through analysis of signal intensity the suitable ratios corresponding to nine primer sets would be confirmed. 4 μL PCR products was mixed with 6 μL of hybridization buffer (5 × SSC, 0.2% SDS, 5% formamide) without heat denaturation treatment, and then transferred to one well on the slide, which was placed in a humidified chamber. The chamber was submerged in a 50 °C water bath, and incubated for 1 h. After removed from the hybridization chamber, the slide was washed in solution A (1 × SSC, 0.2% SDS), solution B (0.2 × SSC) and washing solution C (0.1 × SSC) in sequence for 1 min, then air-dried. Subsequently 1:1500 diluted streptavidin-horseradish peroxidase was incubated in each well on the slide for 30 min at 37 °C, and the slide was washed with PBS-T (0.05% Tween 20) 1 min for three times. Finally, 1:1000 diluted TSACy3 (in 1 × PBS plus 1% BSA) was incubated in each well for 30 min at 37 °C, and the slide was washed according to above step, dried at room temperature. Fluorescent measurements of DNA microarray was generated by scanning the slide on the GenePix 4000B scanner (Axon, USA). The fluorescent signals were quantified using GenePix5.0 software (Axon, USA). All the experiments were done in triple. The cutoff value of each probe was calculated through the mean of the spot intensity in order to determine the signals objectively. DNA microarray was evaluated for sensitivity using a series of 10-fold dilution (106 cfu/ml to 101 cfu/ml) from pure culture. We selected genomic DNA from Salmonella typhi, Shigella spp, Brucella spp, Y. enterocolitica, V. cholera O139, E. coli O157:H7 respectively. Each dilution series was repeated three times. Two following ways were utilized for evaluating sensitivity. One way for S. typhi: each diluted concentration of genomic DNA of S. typhi was diluted with supernatant of stool samples from healthy volunteers, and the other way for genomic DNA of target pathogens in pure culture. We interrogated the difference of two labeling methods, TSA-Cy3 and Cy3 end labeling. PCR products of S. typhi labeled with biotin were diluted by a series of 10-fold dilutions. A parallel dilution series of

Fig. 1. a. Layout of DNA microarray. The name of gene was corresponding to target pathogen. Each probe was spotted as two. b. The typical hybridization results of six species of human pathogenic microorganism and non-target bacteria from pure bacterial cultures. (1) non-target bacteria; (2) Yersinia enterocolitica; (3) Brucella spp.; (4) Shigella spp.; (5) Salmonella typhi; (6) Vibrio cholera non-O139; (7) Vibrio cholera O139; (8) Escherichia coli O157:non-H7; (9) Escherichia coli non-O157:H7; (10) Escherichia coli O157:H7.

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Table 2 Genes targeted for multiplex PCR and DNA microarray hybridization Pathogenic microorganism

Target genea

GenBank accession no.b

Primer namec

Frag. size (bp)

Primer sequence(5′-3′)

Vibrio cholera O139

ctxA

DQ774432

174

LPSgt

U72485

rfbE

S83460

filC

AM228905

Yersinia enterocolitica

Ail

AY004311

Shigella spp

ipaH

DQ448042

Salmonella typhi

vipR

X67785

Brucella spp

BCSP

DQ229169

Internal control

16S rDNA

U00096

ctxA-F ctxA-R ctxA-P LPSgt-F LPSgt-R LPSgt-P rfbE-F rfbE-R rfbE-P filC-F filC-R filC-P ail-F ail-R ail-P ipaH-F ipaH-R ipaH-P vipR-F vipRRvipR-P BCSP-F BCSP-R BCSP-P 16S-F 16S-R 16S-P

CACCCAACATGTTTAACGTTAATG ATCTATCTCTGTAGCCCCTATTAC CATACAGTCCTCATCCAGATGAACAAGAAGTTTCTGCTTTAGGTG CAGATTGTGATATGATAAGAGCGC ATAACAACTGAGATATCAAGCGTC TCGATAAGAAGAGATAAAGATCTGAGTTATCTAAAGATATTTGATTTAATGTTTT AACTATTACTACAGGTGAAGGT TAGCCTATAACGTCATGCCAA AGGCCAAGGATTAGCTGTACATAGGCA GGTGGGATTACTTATCAGGCTAC ATCCACATAAGACTTCGCAGCATC AGATGTAGTATTGAGCGAAACCAAAGCGGCTGCCGCGACATCTTCAATTAC AGGTTCGTTTGCTTATACCCATCAG GCTTAATGCGGAAAGATGGCCCC TTTCTTCTATGGCAGTAATAAGTTTGGTCACGGTGATCTTGA CTCAGTGCCTCTGCGGAGCTTCG GAGAGTTCTGACTTTATCCCG GAAGGCCTTTTCGATAATGATACCGGCGCTCTGCTCTCCC GGTTTCATCATTTCTGGCCTCCG CTCTGCTCCGTCAAGATCTTTTCACC CTTCAATAATGCCAGCAGCTCCAACCCCGAAATAGATA TGGCTCGGTTGCCAATATCAA CGCGCTTGCCTTTCAGGTCTG CTGGCGACGCTTTACCCGGAAACGATCCATA CGCTGGCGGCAGGCCTAACACATGC CGCGGCTGCTGGCACGGAGTTAGCC ACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATATTG

Escherichia coli O157:H7

262

125

162

116

234

337

223

500

a Genetic locus targeted by the described PCR primers and probes: ctxA: toxin sub-unit A, LPSgt: glycosotransferase, rfbE: O antigen for O157 serotype, fliC: H7 flagellar antigen, ail: attachment invasion locus, ipaH: invasion plasmid antigen H, vipR: regulating Vi antigen expression, BCSP: 31-kDa cell surface protein. b Representative GenBank accession number from which the probe sequence was derived. c F, sequence of the forward primer; R, sequence of the reverse primer; P, Oligonucleotide probe.

PCR products labeled with Cy3 was prepared according to the assay described previously (Jin et al., 2006). Oligonucleotide sequence of primers and probes used in this study are shown in Table 2. The specificity of primers and probes was confirmed by 92 reference strains from 18 genera or species. The results indicated that the positive signals emerged at the position of internal probe, while blank control and non-bacterial DNA control showed no signals (Fig. 1-b). Different ratio of the forward primer vs reverse primer (labeled with biotin) was tested in PCR reaction for each amplicon. The results showed that the intensity of the hybridization signal from each probe was the highest while the ratios of the primer pairs for 16S rDNA, ctxA, LPSgt, rfbE, fliC, ail, ipaH, vipR, and BCSP gene were adjusted to be 1:10, 1:10, 1:10, 1:10, 1:5, 1:10, 1:10, 1:5 and 1:5, respectively. The cutoff

value is a proxy to determine hybridization results. The average intensity of signals from negative bacteria and blank control plus 2SD was calculated as cutoff value of probe (data not shown). A total of 92 strains of bacteria from 18 genera or species were tested by DNA microarray assay established as above. The hybridization results indicated that high specificity of hybridization signals was obtained from six species of bacteria. In addition, two serotypes were discriminated between V. cholera O1 and O139 as well as E. coli O157: H7 and non H7. No cross-reacted signals were detected for each target pathogen. Furthermore, positive signal was obtained from internal control probe in each reaction with bacterial genomic DNA, and no signals emerged by the negative control probe in any reaction. We chose S. typhi as target pathogen to compare the method of TSA-Cy3 labeling with the assay of Cy3 end labeling. A series of images

Fig. 2. A series of scanning image obtained from serial dilution of Salmonella typhi PCR products. A: TSA-Cy3 labeling; B: Cy3 end labeling.

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and data corresponding to each concentration were analyzed using GenePix pro 4.0 software (Fig. 2). These results indicated that the method of TSA-Cy3 labeling was ten times more sensitive than the assay of Cy3 end labeling. The sensitivity of DNA microarray was evaluated based on cutoff values, hybridization signals were shown positive for dilution containing as high as 103 cfu/mL of each species of pathogens, which included S. typhi. It was clear that background of genomic DNA in stool samples did not have an affect to the limit of detection. A total of 362 clinical specimens were detected by bacteria culture, API test system and DNA microarray, respectively. Negative and positive controls were included in parallel with each test to avoid false–positive and false–negative results. The results showed that 137 (37.8%) were detected positive of V. cholera O139 by the conventional bacteria culture method, while 142 (39.2%) were positive by using DNA microarray. In addition, 10(2.7%) were detected Shigella spp positive, 4(1.1%) were detected E. coli O157:H7 positive and 6(1.7%) were S. typhi positive by both two detection methods. Other two target pathogens were not detected in all clinical specimens. Furthermore, the results obtained by the appropriate API test system were the same to that of DNA microarray. All the clinical specimens were collected from an epidemic of V. cholera, so it was predicable that pathogenic V. cholera O139 should be the etiological agent in this epidemic, which was confirmed experimentally by the assay described here. The results also showed that 357 (98.6%) specimens identified by DNA microarray were also positive by conventional bacteria culture method. However, DNA microarray was able to detect 5 extra positive specimens that were negative with the conventional method, but were positive with the appropriate API test system, suggesting DNA microarray was more intact than the conventional culture method assay when clinical specimens contained dead or inactive bacteria. 5 mocked double-blind samples were detected by using DNA microarray. The results showed that there were 4 positive samples including 1 Y. enterocolitica, 1 Brucella spp, 2 mixed pathogens (E. coli O157:H7 and Shigella spp; E. coli O157:H7 and S. typhi), and 1 negative samples in 5 mocked samples. In this report, we have established an assay using DNA microarray coupled with multiplex PCR and TSA-Cy3 labeling method for simultaneous identification of six species of human diarrheal pathogenic microorganisms and discriminate V. cholera serotype O139 from O1 based on the present of the LPSgt gene, and specifically detect E. coli O157 and serotype H7 based on the O157-(rfbE) and H7-specific(fliC)

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