Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains

Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, an EvaGreen-based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus.     

A multiplex PCR assay for the detection of five human pathogenic Vibrio species and Plesiomonas

A multiplex PCR (mPCR) assay was established to detect five pathogenic Vibrio species and Plesiomonas shigelloides. Twelve genes were included: ompW, ctxA, rfbN, and wbfR from V. cholerae; tl, tdh, and trh from V. parahaemolyticus; toxR and vmhA from V. mimicus; toxR from V. fluvialis; vvhA from V. vulnificus; and the 23S rRNA gene from P. shigelloides. The specificity of the mPCR assay was 100% for the detection of 136 strains and the limits of detection (LoD) were 12.5–50 pg/reaction. The assay exhibited higher sensitivity than cultivation methods in the detection of APW cultures of 113 diarrhea samples. In the analysis of 369 suspected Vibrio populations from estuarine water samples, the specificity of the mPCR for V. cholerae and V. parahaemolyticus was 100% for both, while the sensitivities were 100% and 96.1%, respectively. The assay can be applied to screen enrichment cultures and suspected colonies from environmental and clinical samples.     

Multiplex PCR for the detection and differentiation of Vibrio parahaemolyticus strains using the groEL,tdh and trh genes

Vibrio parahaemolyticus is a significant cause of human gastrointestinal disorders worldwide, transmitted primarily by ingestion of raw or undercooked contaminated seafood. In this study, a multiplex PCR assay for the detection and differentiation of V. parahaemolyticus strains was developed using primer sets for a species-specific marker, groEL, and two virulence markers, tdh and trh.Multiplex PCR conditions were standardised, and extracted genomic DNA of 70 V. parahaemolyticus strains was used for identification. The sensitivity and efficacy of this method were validated using artificially inoculated shellfish and seawater. The expected sizes of amplicons were 510 bp, 382 bp, and 171 bp for groEL, tdh and trh, respectively. PCR products were sufficiently different in size, and the detection limits of the multiplex PCR for groEL, tdh and trh were each 200 pg DNA. Specific detection and differentiation of virulent from non-virulent strains in shellfish homogenates and seawater was also possible after artificial inoculation with various V. parahaemolyticus strains.This newly developed multiplex PCR is a rapid assay for detection and differentiation of pathogenic V. parahaemolyticus strains, and could be used to prevent disease outbreaks and protect public health by helping the seafood industry maintain a safe shellfish supply.     

CARB-17 Family of β-Lactamases Mediates Intrinsic Resistance to Penicillins in Vibrio parahaemolyticus

Vibrio parahaemolyticus is commonly resistant to ampicillin, yet the mechanisms underlying this phenomenon are not clear. In this study, a novel class A carbenicillin-hydrolyzing β-lactamase (CARB) family of β-lactamases, bla_CARB-17, was identified and found to be responsible for the intrinsic penicillin resistance in V. parahaemolyticus. Importantly, bla_CARB-17-like genes were present in all 293 V. parahaemolyticus genome sequences available in GenBank and detectable in all 91 V. parahaemolyticus food isolates, further confirming the intrinsic nature of this gene.     

Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control

A multiplex polymerase chain reaction (PCR) assay that can simultaneously detect 4 major Vibrio spp., Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae, in the presence of an internal amplification control (IAC) was developed. Species-specific PCR primers were designed based on the gyrB gene for V. alginolyticus, the collagenase gene for V. parahaemolyticus, the vvhA gene for V. vulnificus, and the ompW gene for V. cholerae. Additionally, an IAC primer pair was designed in conserved regions of the bacterial 16S rRNA gene that is used to indicate false-negative results. A multiplex PCR method was developed after optimization of the reaction conditions. The specificity of the PCR was validated by using 83 Vibrio strains and 10 other non-Vibrio bacterial species. The detection limit of the PCR was 10 CFU per tube for V. alginolyticus, V. parahaemolyticus, V. vulnificus, and 10⁵ CFU per tube for V. cholerae in mixed conditions. This method was used to identify 69 suspicious Vibrio isolates, and the results were consistent with physiological and biochemical tests. This multiplex PCR method proved to be rapid, sensitive, and specific. The existence of IAC could successfully eliminate false-negative results for the detection of V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. cholerae.     

Development of real-time PCR assays for specific detection of hmsH,hmsF, hmsR,and irp2 located within the 102-kb pgm locus of Yersinia pestis

Virulent isolates of three pathogenic Yersinia species (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) harbor a 102-kb chromosomal region which encodes elements critical for virulence. A 35-kb high pathogenicity island is contained in this region, is a known virulence determinant, contains irp1 and irp2 iron-regulating genes. An additional segment, the 68-kb high pathogenicity island, contains genetic elements responsible for conferring the Y. pestis pigmentation phenotype on Congo red agar at 28 °C. Collectively, these contiguous segments are referred to as the pigmentation (pgm) locus, the absence of which results in strain attenuation and exemption from CDC Select Agent status. In this study, we developed a set of four real-time PCR assays to detect the presence or absence of multiple virulence genes located within this region. Specifically, we designed TaqMan^® PCR assays to individually detect three hemin storage genes (hmsH, hmsF, and hmsR) which are genetic elements that confer the pigmentation phenotype, as well as the iron-regulating status of 25 Y. pestis isolates (representing 23 different strains), thus establishing a molecular based assay capable of determining the pgm status of candidate Y. pestis isolates. Included in the validation process, was a comparison of these real-time PCR assays and newly developed conventional PCR assays targeting much larger areas of the 102-kb region (including one assay spanning hmsR and hmsF, one spanning hmsH and hsmF, one targeting hmsF, and one targeting irp2). There was high concordance between the conventional and real-time PCR assays for all Y. pestis strains tested. The results from the comparative analysis document the specificity and sensitivity of the real-time PCR assays and further solidify the ostensible benefits of real-time PCR over conventional PCR.     

Genotypes and Virulence Genes in Group B Streptococcus Isolated in the Maternity Hospital,Kuwait

Objective

To characterize group B streptococcus (GBS) isolates obtained from patients at the Maternity Hospital in Kuwait for their genotypes and carriage of virulence genes.

Materials and Methods

A total of 154 GBS isolates were obtained from July 1 to October 31, 2007, from vaginal swabs (n = 95), urine (n = 46), blood (n = 4) and miscellaneous sources (n = 9). Genotypes were obtained by pulsed-field gel electrophoresis (PFGE), following digestion with SmaI or EagI restriction enzymes. PCR was used to screen for the carriage of virulence genes including: surface protein of group B streptococcus (spb1), secreted fibrinogen-binding protein (fbsB), C5a peptidase (scpB), laminin-binding protein (lmb), α− (bca) and β-subunits of the C protein (bac), resistance to protease immunity protein (rib), and phage-associated gene (pag); regulatory protein (dltR), and toxins CAMP factor (cfb), hyaluronidase (hylB) and superoxide dismutase (sodA).

Results

PFGE defined 14 genotypes differentiating isolates with the same serotypes into different genetic backgrounds. All isolates contained genes for virulence factors. However, cfb (99.4%), scpB (88.3%), lmb (88.3%), bca (57.8%), sodA (55.8%) and dltR (53.9%) were the common virulence genes. In total, 144 (90.3%) of the isolates contained 3 or more virulence genes. However, while cfb, lmb and scpB occurred in all genotypes, others occurred in some but not in all genotypes.

Conclusions

GBS isolates obtained at the Maternity Hospital, Kuwait, belonged to diverse genetic backgrounds with the majority carrying multiple virulence genes.Key Words: Group B streptococcus, Genotypes, Pulsed-field gel electrophoresis, Virulence genes     

First Detection of AmpC β-Lactamase blaCMY-2 on a Conjugative IncA/C Plasmid in a Vibrio parahaemolyticus Isolate of Food Origin

Vibrio parahaemolyticus is an important causative agent of gastroenteritis, with the consumption of contaminated seafood being the major transmission route. Resistance to penicillin is common among V. parahaemolyticus strains, whereas cephalosporin resistance remains rare. In an attempt to assess the current prevalence and characteristics of antibiotic resistance of this pathogen in common food samples, a total of 54 (17% of the total samples) V. parahaemolyticus strains were isolated from 318 meat and seafood samples purchased from supermarkets and wet markets in Shenzhen, China, in 2013. These isolates exhibited high-level resistance to ampicillin, yet they were mostly susceptible to other antimicrobials, except for two that were resistant to extended-spectrum cephalosporins. The β-lactamase gene bla_PER-1 was detectable in one strain, V. parahaemolyticus V43, which was resistant to both third- and fourth-generation cephalosporins. Compared to other bla_PER-1-positive V. parahaemolyticus strains reported in our previous studies, strain V43 was found to harbor an ∼200-kb conjugative plasmid carrying genes that were different from the antimicrobial resistance genes reported from the previous studies. The β-lactamase gene bla_CMY-2 was detectable for the first time in another V. parahaemolyticus isolate, V4, which was resistant to third-generation cephalosporins. This bla_CMY-2 gene was shown to be located in an ∼150-kb IncA/C-type conjugative plasmid with a genetic structure consisting of traB-traV-traA-ISEcp1-bla_CMY-2-blc-sugE-encR-orf1-orf2-orf3-orf4-dsbC-traC, which is identical to that of other IncA/C conjugative plasmids in Enterobacteriaceae, albeit with a different size. These findings indicate that the transmission of extended-spectrum-β-lactamase (ESBL) and AmpC β-lactamase genes via conjugative plasmids can mediate the development of extended-spectrum cephalosporin resistance in V. parahaemolyticus, thereby posing a potential threat to public health.     

A real-time multiplex PCR for the identification and typing of Vibrio cholerae

We report the development and validation of a duo-triplex real-time polymerase chain reaction (PCR) for the rapid identification and typing of Vibrio cholerae. The PCR assay targets a species-specific toxR gene present in all strains of V. cholerae and used as a marker for the species wbeO1 and wbfO139, encoding the O1 and O139 somatic antigens, and ctxA, encoding cholera toxin (CT). The two tcpA variants associated with the classical and El-Tor biotypes are used to infer biotype. The assay was evaluated using 178 isolates comprising eight different Vibrio species, including 122 isolates of V. cholerae. The PCR results of 171/178 (96.1%) isolates were concordant with the serotyping, biotyping, and expected CT results. Variants of toxR (n = 3), nonfunctional wbeO1 (n = 1), and CT-negative isolates of V. cholerae O1 (n = 3) were likely explanations for the mismatched results. This duo-triplex real-time PCR is a reproducible and robust assay for the rapid identification and typing of V. cholerae belonging to the highly pathogenic, pandemic lineages.     

A Study on Detection of Pathogenic Listeria monocytogenes in Ovine’s of Kashmir Region Having Abortion or History of Abortion

The present investigation delineates the role of listeric infection in aborted ewes or those with history of abortion from organized farms of Kashmir region. A total of 141 clinical samples was analyzed for the isolation and identification of Listeria species. On analysis four isolates were identified as Listeria monocytogenes, while 24 were non-pathogenic Listeria species. Of the four L. monocytogenes isolates, two were isolated from brain tissue of aborted fetus (BrS10 and BrG36) while one each was isolated from vaginal swab (Vd13) and rectal swab (RS11) of the ewe. An overall isolation rate of 2.83 % was observed for L. monocytogenes and 17.02 % for non-pathogenic Listeria species. Further to reveal the pathogenic potential, the recovered L. monocytogenes isolates were subjected to the battery of in vitro pathogenicity test such as hemolytic activity on Sheep Blood Agar, Phosphatidyl Inositol-Phospholipase C activity on Agar Listeria according to Ottaviani and Agosti medium, multiplex PCR targeting virulence markers genes viz., prfA, plcA, actA, hly, inlC and in vivo chick embryo inoculation test. All the L. monocytogenes isolates recovered in the present study were potentially pathogenic and on comparison, a good correlation was observed among in vitro and in vivo pathogenicity test including multiplex PCR targeting virulence associated genes.     

Intracellular invasion ability of Streptococcus agalactiae among non-invasive isolates from human adults and companion animals in Japan

ObjectiveThis study evaluated the cell invasion ability (CIA) of Streptococcus agalactiae isolates from humans and companion animals and clarified the relationship between CIA populations and their microbiological features.MethodsHuman-origin and companion animal-origin isolates were collected along with host information. We measured CIA using human-lineage colon cancer epithelium (Caco-2) and keratinocyte (HaCaT) cell lines, via virulence-associated gene profiling (bca-rib-bac-lmb-cylE-hylB-pavA-pilB-spb1-srtC1-brpA), capsular genotyping, multilocus sequence typing, and antimicrobial resistance (AMR) phenotyping/genotyping. Significant differences in data regarding CIA into epithelium and keratinocytes and those of isolates from different hosts were assessed. We analyzed the association of CIA populations with the virulence genotypes, capsular genotypes, sequence types/clonal complexes, and AMR phenotypes/genotypes.ResultsA comparative analysis was performed between human (n = 15) and canine (n = 17) non-invasive isolates. There was a difference in CIA data between Caco-2 and HaCaT cells using human and animal isolates. For percent invasion ability into Caco-2 cells, we designated values ≥ 0.1 as high-frequency CIA and values < 0.1 as low-frequency CIA. Fourteen isolates harbored high-frequency and 18 isolates harbored low-frequency strains. There was no association between the high-frequency population and the virulence genotypes, capsular genotypes, sequence types/clonal complexes, and AMR phenotypes/genotypes.ConclusionThis is the first report assessing the invasion ability of S. agalactiae into HaCaT and Caco-2 cells. Our observations suggest that S. agalactiae is more capable of entering Caco-2 rather than HaCaT.     

Triplex PCR assay for the rapid identification of 3 major Vibrio species, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio fluvialis

A triplex PCR assay was developed for the identification of 3 major Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio fluvialis by targeting their haemolysin, haem-utilizing, and central regulatory genes, respectively. This simple, rapid, sensitive, and specific assay using cell lysates from 227 samples established its usefulness in research and epidemiology.     

Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction

Polymerase chain reaction (PCR) protocols were established for specific detection of the tdh and trh genes, the virulence marker genes of Vibrio parahaemolyticus encoding two related hemolysins. The tdh and trh genes are known to have sequence divergence of up to 3 · 3% and 16%, respectively. Attempts were made to find suitable primer pairs and annealing temperatures to detect each gene without fail. DNAs extracted from 36 representative strains of V. parahaemolyticus were used in the initial screening with various combinations of primer pairs and annealing temperatures. The combinations of primer pairs and annealing temperatures selected were then tested with DNAs extracted from 227 more strains of V. parahaemolyticus and from 133 bacterial strains belonging to 40 species other than V. parahaemolyticus. PCR protocols (primer pairs and annealing temperatures) were established that gave identical results to those obtained with the tdh- and trh-specific polynucleotide probes. These protocols established for the tdh and trh genes could detect 400 fg (100 cells) of cellular DNA carrying the respective gene. Spike experiments demonstrated that the sensitivities of the established PCRs were reduced by a factor of 10⁴–10⁵ by an inhibitor(s) present in a normal faecal sample, indicating the need for either DNA extraction or enrichment of the faecal sample in alkaline peptone water for 4 h before the PCR of faecal samples.     

Molecular characteristics of antimicrobial resistance and virulence determinants of Staphylococcus aureus isolates derived from clinical infection and food

Background

Staphylococcus aureus (S. aureus) is an important human etiologic agent. An investigation of the characteristics of common genotypes of S. aureus relating to pathogenicity and antibiotic resistance may provide a foundation to prevent infection.

Methods

This study collected 275 S. aureus isolates from Zhengzhou city in China, including 148 isolates from patient samples and 127 isolates from ready‐to‐eat food samples. Antimicrobial susceptibility testing was performed using the broth dilution method. Molecular characteristics of antimicrobial resistance, virulence, and genotypes were identified by polymerase chain reaction (PCR).

Results

In total, 34.18% (94/275) of S. aureus isolates were MRSA. Compared with food isolates, clinical isolates had significantly higher antibiotic resistance rates, carrying resistance genes such as acc(6′)/aph(2′), aph(3′)‐III, ermA, and ermB and virulence genes such as tetM, sea, seb, pvl, and etb. MRSA‐t030‐agrI‐SCCmecIII and MSSA‐t002‐agrII were the most common strain types among clinical strains, and MRSA‐t002‐agrII‐SCCmecIII and MSSA‐t002‐agrII were the most common strain types among food strains. Additionally, some strains in the agr group were also spa type‐specific, suggesting that there may be phenotypic consistency.

Conclusion

Clinical isolates contained higher numbers of resistance genes and demonstrated higher antibiotic resistance, while 2 source strains exhibited high toxicity. These results indicate that bacteria with different origins may have undergone different evolutionary processes. As resistance and virulence factors in food bacteria can be transmitted to humans, food handlers should strictly follow hygienic measures during food production to ensure the safety of human consumers.

     

A potential role of aminoglycoside resistance in endemic occurrence of Pseudomonas aeruginosa strains in lower airways of mechanically ventilated patients

Altogether, 98 Pseudomonas aeruginosa isolates from a 5-bed intensive care unit were fingerprinted with pulsed-field gel electrophoresis and tested for aminoglycoside resistance genes aac(6′)-Ib, aac(3″)-IIa, ant(2″)-Ia, armA, rmtA, and rmtB and integrons and virulence genes/operons phzI, phzII, phzM, phzS, apr, lasB, plcH, plcN, pilA, algD, toxA, exoS, exoT, exoY, and exoU. Two major clusters were identified (49 and 19 isolates), harbouring aac(6′)-Ib, bla_PSE-1, and ant(3″)-Ia genes or ant(2″)-Ia gene, respectively, on a class I integron. Most virulence genes except for exoU and pilA were found. Only 1 isolate of the minor cluster (8 isolates) and 1 of the 22 sporadic isolates carried integrons (without gene cassettes); virulence profile was highly variable. Comparing the resistance and virulence patterns of endemic and sporadic isolates suggests that integron-borne aminoglycoside resistance is more closely associated with the frequency than virulence. Consequently, aminoglycoside usage may have played a role in maintenance of the endemic clones.     

Virulence factors of Proteus mirabilis clinical isolates carrying blaKPC-2 and blaNDM-1 and first report blaOXA-10 in Brazil

IntroductionProteus mirabilis is one of the main pathogens that cause urinary tract infections. Therefore, the aim of this study was to analyze and compare the genetic profile of 36 clinical isolates of P. mirabilis that carry and do not carry the bla_KPC and bla_NDM gene with respect to virulence factors (mrpG, pmfA, ucaA, nrpG and pbtA) and antimicrobial resistance (bla_VIM, bla_IMP, bla_SPM, bla_GES, bla_OXA-23-like, bla_OXA-48-like, bla_OXA-58-like and bla_OXA-10-like).MethodsThe virulence and resistance genes were investigated by using PCR and sequencing.ResultsERIC-PCR typing showed that the isolates showed multiclonal dissemination and high genetic variability. The gene that was most found bla_OXA-10-like (n = 18), followed by bla_KPC (n = 10) and bla_NDM (n = 8). To our knowledge, this is the first report of bla_OXA-10 in P. mirabilis in Brazil, as well as the first report of the occurrence of P. mirabilis co-carrying bla_OXA-10/bla_KPC and bla_OXA-10/bla_NDM. The bla_NDM or bla_KPC carrier isolates showed important virulence genes, such as ucaA (n = 8/44.4%), pbtA (n = 10/55.5%) and nrpG (n = 2/11.1%). However, in general, the non-carrier isolates of bla_KPC and bla_NDM showed a greater number of virulence genes when compared to the carrier group.ConclusionClinical isolates of P. mirabilis, in addition to being multi-drug resistant, presented efficient virulence factors that can establish infection outside the gastrointestinal tract.     

Purification,characterization, and pathogenicity of urease produced by Vibrio parahaemolyticus

Vibrio parahaemolyticus is a halophilic bacterium associated with gastroenteritis and traveller's diarrhea. The urease-positive, Kanagawa-negative V. parahaemolyticus had been isolated from patients and the environment in the Pacific Northwest, first reported by Kelly at al. (5). Recently, we purified the urease produced by a clinical isolated of V. parahaemolyticus, and its characterization and pathogenicity has been studied. The urease isolation procedure included a water extraction and anion exchange chromatography. The molecular weight for the native enzyme was 275 KDa, and the three subunits of 85 KDa, 59 KDa, and 33 KDa were determined. The isoelectric focusing of urease was 5.2. The purified urease also can cause intestinal fluid accumulation and demonstrate a positive result in the suckling mouse test. These results suggested that the urease produced by V. parahaemolyticus may be another important indicator for the pathogenesis of the bacteria. © 1996 Wiley-Liss, Inc.     

The prevalence of aminoglycoside-modifying enzymes among coagulase negative staphylococci in Iranian pediatric patients

In spite of widespread emergence of aminoglycoside resistance, these drugs are still used in the treatment of staphylococcal infections. This study aimed to investigate the distribution of aminoglycoside resistance and genes encoding aminoglycoside – modifying enzymes (AMEs) as well as Staphylococcal Cassette Chromosome mec (SCCmec) type in coagulase negative staphylococci (CoNS) in pediatric patients. Totally, 93 CoNS isolates were examined for susceptibility to aminoglycosides using disk diffusion and/or E-test methods. AMEs genes and SCCmec types were detected using multiplex PCR. Strain typing was performed using repetitive extragenic palindromic (REP) – PCR assay. The non-susceptibility rates to kanamycin, tobramycin, gentamicin, amikacin and netilmicin were 73%, 59%, 49.5%, 16% and 7.5%, respectively. aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia and aph(3′)-IIIa were encountered in 56 (60.2%), 38 (40.8%) and 18 (19.3%) isolates, respectively. In aac(6′)-Ie-aph(2″)-Ia- positive isolates, the non- susceptibility rates to kanamycin, gentamicin, tobramycin, amikacin and netilmicin were 83%, 74%, 73%, 49% and 43%, respectively. SCCmec types included type IV (n = 31), I (n = 17), II (n = 5), III (n = 4), and V (n = 2). Three isolates had two types; I + III (n = 2) and III + IV (n = 1) whereas 11 isolates were non-typeable. AMEs genes carriers were distributed frequently into type IV. We found diverse fingerprint patterns among our isolates. In conclusion, there was a strong correlation between alarming rate of aminoglycoside resistance and methicillin resistance. Discordances between phenotypic and genotypic detection of aminoglycoside resistance were discernible. AMEs genes might be related to SCCmec types.     

Rapid detection of Vibrio parahaemolyticus using magnetic nanobead-based immunoseparation and quantum dot-based immunofluorescence

In recent years, the scale of population exposure and food poisoning caused by Vibrio parahaemolyticus (V. parahaemolyticus) has shown a significant upward trend, becoming one of the primary food-borne pathogens. Herein, we developed a rapid and sensitive detection of V. parahaemolyticus by integrating the technology of magnetic nanobeads (MBs) based immunoseparation (IMS) with quantum dots (QDs) based immunofluorescence. Firstly, specific rabbit polyclone IgG antibodies (IgG) and chicken egg yolk antibodies (IgY) of V. parahaemolyticus were prepared. Then two sizes of MBs (1 μm; 180 nm) were coupled with IgG to form immuno-MB (IMB) capture probes for evaluating the effect of different sizes on the detection efficiency. For QDs, they were conjugated with IgY to form fluorescent reporting probes. In the process of detection, IMB probes were used to separate V. parahaemolyticus and then these complexes were labeled by QD probes on the principle of double antibody sandwich. The fluorescence intensity of the IMB-V. parahaemolyticus-QD complexes was measured by a fluorescence spectrophotometer. The detection method takes 150 min with a detection limit of 10² cfu mL⁻¹ ranging from 10² to 10⁶ cfu mL⁻¹ and it has been shown to work satisfactorily in real food samples.

IMB probe and QD probe were used as separation and fluorescent label to measure the content of V. parahaemolyticus. The detection method with a detection limit of 10² cfu mL⁻¹ has been shown to work satisfactorily in real food samples.