Drug resistance and genomic characteristics of Salmonella enterica serovar London from clinical and food sources in Hangzhou City from 2017 to 2021 / 中华预防医学杂志

Objective: To analyze the drug resistance and genomic characteristics of Salmonella enterica serovar London isolated from clinical and food sources in Hangzhou City from 2017 to 2021. Methods: A total of 91 Salmonella enterica serovar London strains isolated from Hangzhou City from 2017 to 2021 were analyzed for drug susceptibility, pulsed field gel electrophoresis (PFGE) typing and whole genome sequencing. Multilocus sequence typing (MLST), core genome multilocus sequence typing (cgMLST) and detection of drug resistance genes were performed by using the sequencing data. Phylogenetic analysis was conducted to compare the 91 genomes from Hangzhou City with 347 genomes from public databases. Results: No significant difference in the drug resistance rate was observed between clinical strains and food strains to 18 drugs in Hangzhou City(all P>0.05), and the multidrug resistance (MDR) rate was 75.8% (69/91). Most strains were resistant to 7 drug classes simultaneously. One strain was resistant to Polymyxin E as well as positive for mcr-1.1, and 50.5% (46/91) of the strains were resistant to Azithromycin and were positive for mph(A). All 91 Salmonella enterica serovar London strains were ST155, which were subdivided into 44 molecular types by PFGE and 82 types by cgMLST. Phylogenetic analysis showed that most strains from Hangzhou City (83/91) were clustered together, and a small number of human isolates from Europe, North America and pork isolates from Hubei and Shenzhen were mixed in the cluster. Other strains from Hangzhou City (8/91) were closely related to strains from Europe, America and Southeast Asia. Strains isolated from pork were the most closely related to clinical strains. Conclusion: The epidemic of Salmonella enterica serovar London in Hangzhou City is mainly caused by the spread of ST155 strains, which is mainly transmitted locally. At the same time, cross-region transmission to Europe, North America, Southeast Asia, and other provinces and cities in China may also occur. There is no significant difference in the drug resistance rate between clinical strains and food strains, and a high level of MDR is found in the strains. Clinical infection of Salmonella enterica serovar London may be closely related to pork consumption in Hangzhou City.

Study on the changes of blood glucose in hemodialysis patients with diabetes

SUMMARY OBJECTIVE: The aims of this study were to observe the regularity of blood glucose changes in hemodialysis patients with diabetes, time of onset of hypoglycemia and blood glucose level during dialysis, and to explore the sensitive early warning indicators of hypoglycemia in dialysis patients. BACKGROUND: Diabetes patients have a high incidence of hypoglycemia during hemodialysis. METHODS: A total of 124 maintenance hemodialysis patients with diabetes were selected for this study. Before dialysis, one, two, and three h after dialysis, and when hypoglycemia symptoms occurred, the blood glucose changes were monitored, the blood glucose drop range was observed when hypoglycemia symptoms occurred, and the correlation between the two was analyzed. RESULTS: After the start of the dialysis, the patient's blood glucose showed a downward trend. The symptoms of hypoglycemia were most obvious within one-two hours, with an incidence rate of 57.9%. When the blood glucose drop percentage reached 37.7%, the specificity and sensitivity of early warning hypoglycemia symptoms were 84.6 and 73%, respectively. CONCLUSIONS: For hemodialysis patients with diabetes, attention should be paid to the symptoms of hypoglycemia during dialysis, and blood glucose should be monitored before dialysis and after 1-2 h of dialysis. If the blood glucose drop percentage is greater than 37.7%, the timely measures should be taken.

Analysis of infection status and pathogenic features of human metapneumovirus among children in Hangzhou between year 2009 and 2011 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the infection status and pathogenic features of human metapneumovirus (hMPV) among children with acute respiratory tract infection in Hangzhou.</p><p><b>METHODS</b>A total of 372 children less than 14 years old with acute respiratory tract infections were recruited as subjects from the pediatric clinic or intensive care unit (ICU) of 3 hospitals in Hangzhou during November 2009 to January 2010, and November 2010 to January 2011. A total of 372 specimens were collected, including 351 respiratory swab, 9 nasopharyngeal aspirate material, 8 endotracheal aspirate material and 4 sputum. The total nucleic acid was then extracted from the specimens, and the nucleoprotein (N) gene of hMPV was amplified by RT-PCR, whose positive products were sequenced and analyzed. Africa green monkey kidney cells (Vero-E6) were applied to culture hMPV among the positive samples; meanwhile fluorescence quantitative RT-PCR was adopted to test other respiratory virus infection.</p><p><b>RESULTS</b>Out of 372 patients, 42 (11.2%) were positive for N gene of hMPV. The positive rate of hMPV among boys was 11.5% (26/226), and correspondingly 10.9% (16/146) among girls. The difference showed no statistical significance (χ(2) = 0.026, P > 0.05). The youngest patient was only 2 month-old and the eldest patient was 14 years old. The median of the patients' age was 24 months. Fifteen positive samples amplified by RT-PCR were sequenced, and all turned out to be subtype B1; whose similarity to GD165 found in Guangdong was 98.1% - 99.5% and similarity to BJ1897 in Beijing was 87.8% - 89.2%. The co-infection rate between hMPV and other respiratory virus was 45.2% (19/42); most of which was between hMPV and respiratory syncytial virus, whose rate at 26.1% (11/42).</p><p><b>CONCLUSION</b>hMPV was the single genotype relevant with the acute respiratory tract infection disease among children in Hangzhou district; however, the co-infection with other respiratory virus did exist.</p>

Molecular characteristics and antibiotic resistances of Vibrio cholerae O1 isolates in Hangzhou in 2009 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the molecular characteristics and antibiotic resistances of Vibrio cholerae (V. cholerae) O1 isolates in Hangzhou in 2009.</p><p><b>METHODS</b>The virulence genes ctxA and tcpA of the thirty V. cholerae O1 isolates from 7 counties and districts of Hangzhou were detected by PCR. Pulsed-field gel electrophoresis (PFGE) was performed for molecular typing and similarity analysis. Antibiotic resistances of these isolates were measured by the Kirby-Bauer method.</p><p><b>RESULTS</b>Virulence gene analysis showed that 80.00% (24/30) of the genotype in V. cholerae isolates was ctxA- and tcpA+, 13.33% (4/30) was ctxA- and tcpA-, and 6.67% (2/30) was ctxA+ and tcpA+. Twenty-seven isolates tested were typed into 11 PFGE patterns (P1-P11). Twenty-three isolates with genotype ctxA- and tcpA+ were clustered into 7 PFGE patterns (P1-P7, termed P1-like cluster) with the similarity to be equal or greater than 91.4%, and 56.52%(13/23) of them belong to P1. 7 isolates with very high similarity (97.6%), belonging to P1 (6 isolates), and P2 (1 isolate), respectively, were collected from one foodborne disease outbreak. The resistant rates of the 24 isolates with genotype ctxA- and tcpA+ to ampicillin, tobramycin and amikacin were 20.83% (5/24), 4.17% (1/24) and 4.17% (1/24), respectively.</p><p><b>CONCLUSION</b>The genotype of the epidemic strains of V. cholerae O1 isolates in Hangzhou in 2009 with high similarity was ctxA- and tcpA+; The level of drug resistances of this kind of V. cholerae O1 isolates were not high.</p>

Multiplex PCR assay for dissemination and diversity of extended-spectrum beta-lactamase genes in Shigella isolates / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a rapid and simple multiplex polymerase chain reaction (PCR) method which discriminates extended-spectrum beta-lactamases (ESBLs) genes in sporadic Shigella isolates from 1998 to 2007 in Hangzhou city, China.</p><p><b>METHODS</b>After ESBLs screening according to the Clinical and Laboratory Standards Institute (CLSI) method, CTX-M, TEM, SHV and OXA-1 encoding genes were detected by using a multiplex PCR method, and the results were verified by 8 single gene PCR amplification.</p><p><b>RESULTS</b>Seventeen isolates harbored ESBLs genes among 195 Shigella isolates (8.72%). Genes encoding CTX-M (17 strains), TEM (2 strains), OXA-1 (10 strains) and SHV (0 strains) were discriminated with multiplex PCR analysis, which coincided with eight single gene PCR analysis at 94.12%.</p><p><b>CONCLUSION</b>Multiplex PCR should be a suitable tool for initial rapid screening and discriminating ESBLs genes in Shigella isolates. With similar trend of national surveillance data, the proportion of sporadic Shigella isolates harbouring ESBLs genes might probably be on increase.</p>

Heterozygous genotypes and molecular characteristics of Organophosphorus resistance associated esterase B2 genes of Culex pipiens complex / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the heterozygous genotype and molecular characteristics of Organophosphorus resistance associated with heterozygous Estbeta2 of esterase B2 gene from natural population of Culex pipiens complex.</p><p><b>METHODS</b>Genomic DNA was extracted from natural populations of Culex pipiens complex in Hangzhou. The PCR-restriction fragment length polymorphism (PCR-RFLP) assay was applied to type the resistance associated esterase gene. Estbeta2 of esterase B2 gene was identified by PCR-RFLP, and the genotyping for heterozygous Estbeta2 was carried out after restriction enzyme digesting by Bfm I endonuclease.</p><p><b>RESULTS</b>The DNA was isolated from 207 Culex pipiens respectively, while 156 PCR samples showed positive and the positive rate was 75.36% (156/207). The PCR-RFLP assay of esterase B2 gene revealed that the Estbeta2 was accounted about 28.20% (44/156) in 156 positive samples. There were two genotypes identified, namely homozygous Estbeta2 (90.90%, 30/33) and heterozygous Estbeta2 (9%, 3/33), heterozygous Estbeta2 was in existence of a hybrid form as which combined with Estbeta2 and a subtype (Estbeta2/Estbeta2(1)).</p><p><b>CONCLUSION</b>Heterozygous Estbeta2 of Organophosphorus resistance associated with esterase genotype was determined in natural population of Culex pipiens, and a genotyping method was established.</p>

mRNA expression levels of p53 and DNA damage and repair genes in peripheral blood lymphocytes of benzene-exposed workers / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the mRNA expression levels of p53-mediating DNA damage and repair genes in the peripheral blood lymphocytes of workers and their exposures to benzene in their working environment.</p><p><b>METHODS</b>The mRNA expression levels of p53 and related genes were determined by SYBR Green I chimeric fluorescence quantitative real-time RT-PCR analysis in peripheral blood lymphocytes of 72 workers, who were classified into group A (46 direct exposure to benzene) and group B (26 indirect exposure to benzene) based on their positions, and 29 controls. The differences of gene expression levels were analyzed by software REST 2005. Meanwhile, the peripheral blood leukocytes, hemoglobin and platelet of workers and controls were counted. Benzene content was measured in the samples of toluene, used as raw material, and spraying agents and benzene, toluene and xylene concentrations in the air of workplaces were monitored.</p><p><b>RESULTS</b>There were no significant differences in the mRNA expression levels of p53, Ku80, Ape1 and Mdm-2 between group A or group B and control group (P > 0.05). The expression up-regulation of p21 mRNA was found, but without significant difference (P > 0.05). However, the mRNA expression levels of Rad51, Bcl-2, Bax, Xpa and Xpc in group A and Rad51 in group B were downregulated significantly (P < 0.05 or P < 0.01). Moreover, both the counts of white blood cell, hemoglobin and platelet in group A were (4.93 +/- 1.27) x 10(9)/L, (123.97 +/- 11.80) g/L and (124.02 +/- 41.22) x 10(9)/L respectively and platelet in group B (135.80 +/- 39.44) x 10(9)/L were significantly lower than in control group (P < 0.05 or P < 0.01).</p><p><b>CONCLUSION</b>The mRNA expression levels of some p53-mediating DNA damage and repair genes are downregulated in the workers chronically exposed to low benzene concentration. The working environment impacts on health of group A workers are greater than the ones of group B.</p>

Establishing a single-tube fluorescent bidirectional PCR method to detect the 609C/T polymorphism of NQO1 gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a single-tube fluorescent bidirectional PCR method to detect the 609C/T polymorphism of NAD(P)H: quinone oxidoreductase 1 (NQO1) gene.</p><p><b>METHODS</b>Two primers of NQO1 gene C609T locus were designed. Using these primers, a SYBR Green I fluorescent bidirectional PCR, combined with melting curve analysis of the PCR products, were optimized to differentiate the 609C/T polymorphisms in 191 samples of human genomic DNA. The accuracy of the fluorescent bidirectional PCR was validated by the classical method of PCR-restriction fragment length polymorphism(RFLP) in 62 of these 191 samples.</p><p><b>RESULTS</b>In the 62 samples, the genotypes determined by the fluorescent bidirectional PCR were 100% consistent with the ones by the PCR-RFLP. The frequencies of genotypes of homozygous wild-type (CC), heterozygous (CT), and homozygous mutant (TT) were 28%, 50%, and 22%, respectively, in the 191 samples.</p><p><b>CONCLUSION</b>The single-tube fluorescent bidirectional PCR method established here provides a simple, rapid, accurate and inexpensive assay to determine the 609C/T polymorphism of NQO1 gene. The assay is suitable to detect the single nucleotide polymorphism in large-scale samples.</p>

Preparation of armored RNA containing M gene of influenza H3N2 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare the armored RNA containing M gene of influenza H3N2.</p><p><b>METHODS</b>The vector pAR-1 was constructed from expression vector pET30b in which the bacteriophage MS2 DNA fragment, containing the genes for maturase and coat protein and the pac site, was inserted. The M gene fragment of influenza A was inserted into the HindIII site downstream of the pac site on the pAR-1, which formed a new recombinant plasmid pAR-2. After the prokaryotic expression was carried out, armored RNA AR-2 containing M gene was obtained. AR-2 was purified, and then was quantified by real time RT-PCR. Moreover, the stability of AR-2 was checked.</p><p><b>RESULTS</b>AR-2 was expressed successfully. AR-2 remained stable under various storage environments. Approximately 8.9 x 10(11) copies of AR-2 particles can be purified from one milliliter of culture.</p><p><b>CONCLUSION</b>It showed that AR-2 was stable and RNase-resistant, which, as a virus surrogate, would be used as RT-PCR standards, controls and training or proficiency samples.</p>

Multiplex real-time PCR detecting Salmonella, Shigella and diarrheagenic Escherichia coli / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a multiplex real-time PCR for the detection of Salmonella invasion protein A gene (invA), enterotoxigenic Escherichia coli (ETEC) heat-labile I enterotoxin gene (elt), and Shigella or enteroinvasive E. coli (EIEC) invasive plasmid antigen H gene (ipaH).</p><p><b>METHODS</b>Under the optimized reaction conditions of the multiplex real-time PCR, invA, elt, and ipaH were determined in 10-fold series of dilution of DNA extracted from Salmonella enterica serovar Typhimurium, ETEC 44815 strain and Shigella F301 strain. The three genes were examined in 90 fecal samples from diarrhea patients using the multiplex real-time PCR. When PCR-positive samples were found, the target strains were isolated and identified.</p><p><b>RESULTS</b>The detectable concentration for this multiplex real-time PCR was 10 CFU/microl for Shigella F301 strain, 10(2) CFU/microl for S. enterica serovar Typhimurium and ETEC 44815 strain, respectively. Out of 90 fecal samples from diarrhea patients, thirteen were found positive for elt gene (14.4%), and five were found positive for ipaH gene (5.6%). Three E. coli strains positive for elt gene and four E. coli strains positive for ipaH gene were isolated successfully from the PCR-positive samples mentioned above. The detection of invA, elt and ipaH genes was completed in 10 h, which included an enrichment period of 6 h.</p><p><b>CONCLUSION</b>The multiplex real-time PCR assay can detect invA, elt, ipaH simultaneously in a single reaction, moreover, it can detect for virulence genes in strains of Salmonella, ETEC, and Shigella or EIEC and screen these pathogens in fecal specimens from patients with diarrhea with a high specificity.</p>

Genotypes, allele frequencies and dynamic distribution on resistance-associated esterase genes of Culex pipiens complex in Hangzhou / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the genotypes , allele frequencies and dynamic distribution on resistance associated esterase genes of Culex pipiens complex in Hangzhou.</p><p><b>METHODS</b>The PCR-restriction fragment length polymorphism (PCR-RFLP) assay was applied to type the resistance associated esterase genes, and dynamic surveillance on frequencies of the resistance associated esterase gene of natural population of Culex pipiens complex in Hangzhou during 2003-2005, and phenotype of the resistance associated esterase genes were detected by esterase starch gel electrophoresis technique.</p><p><b>RESULTS</b>The PCR-RFLP assay of esterase allele genes for three consecutive years disclosed four esterase genotypes, namely, the world-wide highly active homozygous Est beta 1(1) (50%-54%), homozygous Est beta 2 (29%-34%), heterozygous Est beta 1(1)/beta 2 (5%-10%) and Est beta N (3.13%) of a new homozygous genotype. The research of the resistance associated esterase genes phenotype in natural population of Culex pipiens complex in Hangzhou in 2005 with esterase starch gel electrophoresis technique revealed four major types, namely, Est beta 1(1) (61%), Est alpha 2/beta 2 (12%), Est alpha 8/beta 8 (7%) and sensitive phenotype (29%).</p><p><b>CONCLUSION</b>There should be various resistance associated esterase genotypes in natural population of Culex pipiens complex in Hangzhou. During the period of 2003-2005, Est beta 1(1) was the major type; Est alpha 2/beta 2 was the second. Est beta N was a new esterase genotype detected in 2005 only with a mere percentage of 3.13%. As for its resistance to the new insecticide, a follow-up study should be needed. The molecular typing of the amplified esterase gene should be consistent with the resistance associated esterase genes phenotype.</p>

PFGE of Shigella flexneri 4c isolates from food-poisoning outbreaks and sporadic diarrhea patients / 中华预防医学杂志

<p><b>OBJECTIVE</b>To know the molecular characteristic of Shigella flexneri 4c isolates from patients in two food-poisoning outbreaks and one sporadic diarrhea case in Hangzhou, China.</p><p><b>METHODS</b>S. flexneri isolates from patients in two food-poisoning outbreaks (outbreak 1 and outbreak 2, n = 13 and n = 12, respectively) and one sporadic diarrhea patient (n = 1) in Hangzhou during 2003 and 2005 were serotyped. Antibiotic resistances of these isolates were measured by the Kirby-Bauer method. Invasive plasmid antigen gene ipaH was examined by PCR. Pulse field gel electrophoresis (PFGE) was performed for molecular typing.</p><p><b>RESULTS</b>In outbreak 1, all 13 isolates were S. flexneri 4c, of them 6 isolates tested were quite different in PFGE patterns with dice coefficient from 0.78 to 0.92. In outbreak 2, 10 isolates were S. flexneri 4c and 2 isolates were S. flexneri X, however their PFGE patterns were almost identical (dice coefficient > 0.8). Compared to the two outbreaks isolates, the sporadic isolate was demonstrated with a distinct PFGE pattern (dice coefficient < 0.8). The antibiotic resistance patterns with 14 kinds of antibiotics had a little difference among the isolates from outbreak 1, outbreak 2 and sporadic diarrhea patient, but the same pattern was found among 10 isolates of S. flexneri 4c and 2 isolates of S. flexneri X from outbreak 2.</p><p><b>CONCLUSIONS</b>PFGE might distinguish the isolates from these two outbreaks and the sporadic diarrhea patient. Some differences in PFGE patterns, serotypes and antibiotic resistance patterns might occur among S. flexneri 4c isolates during an outbreak.</p>

Molecular typing on Vibrio parahaemolyticus isolates from Hangzhou, China, during 2000-2002 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the molecular epidemiology of Vibrio parahaemolyticus isolates from clinical and environmental samples collected in Hangzhou area during 2000 and 2002.</p><p><b>METHODS</b>V. parahaemolyticus isolates from food-poisoning, sporadic diarrhea patients and seafood in market in Hangzhou during 2000 and 2002 were serotyped. From clinical isolates of serotype O3:K6 and environmental isolates of serotype O3:KUT, virulence genes, tdh and trh, were examined by PCR. Molecular typings [including ribotyping, random amplified polymorphic DNA analysis (RAPD), enterobacterial repetitive intergenic consensus PCR (ERIC-PCR)], were also performed.</p><p><b>RESULTS</b>Among 13 food-poisoning outbreaks caused by V. parahaemolyticus, O3:K6 strains with tdh positive and trh negative were detected in 11 episodes (84.6%), and O4:K8 strains with tdh positive and trh negative were responsible for other 2 outbreaks (15.4%). In 34 V. parahaemolyticus isolates from sporadic diarrhea patients, O3:K6, O4:K8, and O1:KUT strains with tdh positive and trh negative were detected with proportions of 26.5% (9/34), 17.6% (6/34), and 38.2% (13/34), respectively, and other 6 strains were not able to be serotyped. Of 64 isolates from seafood in which both tdh and trh were negative except trh was positive in one O1:KUT strain, 37 belonged to 7 serotypes except O3:K6 or O4:K8, and 9 were not able to be serotyped. The fingprintings of ribotyping, ERIC-PCR, and RAPD showed that almost all of O3:K6 isolates from food-poisoning and sporadic diarrhea patients were genetically close to each other and there were obviously genetical difference between O3:K6 isolates from clinic and O3:KUT isolates from environment.</p><p><b>CONCLUSION</b>There was a distinct serotype distribution between V. parahaemolyticus isolates from clinic patients and seafood. One group of close related V. parahaemolyticus O3:K6 strains with tdh positive and trh negative seemed to be prevailing in Hangzhou in those years.</p>

Detection of severe acute respiratory syndrome probable patients' virus RNA in Hangzhou by using a two loci and a modified nested real-time reverse transcription polymerase chain reaction / 中华预防医学杂志

<p><b>OBJECTIVE</b>To detect the RNA of severe acute respiratory syndrome virus (SARS-CoV) by using reverse transcription polymerase chain reaction (RT-PCR) targeted for a two loci and a modified nested real-time RT-PCR as to improving the reliability and sensitivity of tests.</p><p><b>METHODS</b>A nested RT-PCR was used for detecting one fragment of SARS-CoV RNA in oropharyngeal swabs from 3 SARS probable patients, 4 SARS suspect patients and other 27 patients with fever in Hangzhou, and the nested RT-PCR product from one SARS probable patient was sequenced. Meanwhile in these 3 SARS probable patients, other three RT-PCR methods, including a hemi-nested RT-PCR targeted for another fragment of SARS-CoV RNA, a real-time RT-PCR and a modified nested real-time RT-PCR, were employed to detect SARS-CoV RNA.</p><p><b>RESULTS</b>Two positives were found in the 3 SARS probable patients, and none positive in 4 SARS suspect patients and other 27 patients with fever, using the nested RT-PCR. The sequence of the nested RT-PCR product from one SARS probable patient was identified with the counterpart of SARS-CoV genomes published in public database. The results of the hemi-nested RT-PCR, the real-time RT-PCR and the modified nested real-time RT-PCR in the 3 SARS patients were consistent with the one of the nested RT-PCR. During detecting specimen with low copies of RNA, a weak positive signal was produced after about 35 cycles in the real-time RT-PCR, but a strong positive signal was found only after 10 cycles in the modified nested real-time RT-PCR.</p><p><b>CONCLUSION</b>It might improve the reliability of test by employing RT-PCR targeted for two or more fragments in SARS-CoV genome. The modified nested real-time RT-PCR might have higher sensitivity than the routine real-time RT-PCR.</p>

Characteristics of virulence gene in Vibrio parahaemolyticus strains isolated from clinical patients and environment in Hangzhou, China / 中华预防医学杂志

<p><b>OBJECTIVES</b>To investigate the characteristics of virulence gene in Vibrio parahaemolyticus strains isolated from clinical patients and environment in Hangzhou, China.</p><p><b>METHODS</b>Thermostable direct hemolysin gene (tdh) and thermostable direct hemolysin-related hemolysin gene (trh) were determined in a total of 174 strains of V. parahaemolyticus isolated from patients and environment (seafood) in Hangzhou area by PCR.</p><p><b>RESULTS</b>The tdh was found in 92 out of 94 V. parahaemolyticus strains from food poisoning patients and in 33 out of 34 strains from sporadic diarrhea patients, and trh was not detected in all above clinical strains. Meanwhile the tdh was negative in all V. parahaemolyticus strains from environment, and the trh was also negative except one strain with urease activity. All strains with trh negative had no the activity of urease.</p><p><b>CONCLUSIONS</b>The V. parahaemolyticus strains from food poisoning patients and sporadic diarrhea patients are tdh positive and trh negative. The V. parahaemolyticus strains with tdh negative and almost trh positive in environment might be a potential pathogen in Hangzhou.</p>

Development of Multiplex Real-time PCR for Detection of Toxigenic Vibrio cholerae and Virbio parahaemolyticus / 微生物学通报

A multiplex real-time PCR was developed to detect ctxA of Vibrio cholerae, gyrB and tdh of Vibrio parahaemolyticus simultaneously. The multiplex real-time PCR were evalidated by detection for the three genes in 47 toxigenic V. cholerae O1 and O139 strains (ctxA+; O1=3, O139=44), 25 non-toxigenic V. cholerae strains (ctxA-; O1=12, O139=6, non-O1 and non-O139=7), 116 V. parahaemolyticus strains with or without tdh (73 or 43) and 9 other bacteria strains. The specificity and sensitivity of the multiplex real-time PCR in detection for the ctxA and the tdh genes in the strains tested were both 100.0%, compared to the results by routine PCRs. In the detection for V. parahaemolyticus specific gyrB using the multiplex real-time PCR, all of 116 V. parahaemolyticus strains were positive, and 9 other strains and 72 V. cholerae strains were all negative. The multiplex real-time PCR is a sensitive, specific and quick assay not only for detecting virulence genes of V. cholerae and V. parahaemolyticus but also for identifying V. parahaemolyticus at species level. In addition, two real-time PCRs for detection of V. parahaemolyticus virulence genes trh1 and trh2 were also developed.