Development of Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay to Detect Species-Specific tlh and Pathogenic trh and tdh Genes of Vibrio parahaemolyticus
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Vibrio parahaemolyticus causes severe gastroenteritis in humans after consuming contaminated raw or undercooked seafood. A species-specific marker, the thermolabile hemolysin (tlh) gene, and two pathogenic markers, thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes, have been used to identify V. parahaemolyticus and determine its pathogenicity using both PCR and qPCR assays. To enable testing in field conditions with limited resources, this study aimed to develop a simple and rapid method to detect the species-specific (tlh) and pathogenic (trh and tdh) genes of V. parahaemolyticus using multienzyme isothermal rapid amplification (MIRA) combined with a lateral-flow dipstick (LFD). The amplification of the tlh, trh, and tdh genes could be completed within 20 min at temperatures ranging from 30 to 45 °C (p < 0.05). The test yielded positive results for V. parahaemolyticus but produced negative results for nine Vibrio species and eighteen foodborne pathogenic bacterial species. MIRA-LFD could detect 10 fg of DNA and 2 colony-forming units (CFU) of V. parahaemolyticus per reaction, demonstrating a sensitivity level comparable to that of qPCR, which can detect 10 fg of DNA and 2 CFU per reaction. Both MIRA-LFD and qPCR detected seven tlh-positive results from thirty-six oyster samples, whereas one positive result was obtained using the PCR assay. No positive results for the trh and tdh genes were obtained from any oyster samples using MIRA-LFD, PCR, and qPCR. This study suggests that MIRA-LFD is a simple and rapid method to detect species-specific and pathogenic genes of V. parahaemolyticus with high sensitivity.Keywords:
Thermolabile
Hemolysin
A number of DNA-based diagnostic tools have been developed for the detection of Vibrio parahaemolyticus in seafood. However, the loop-mediated isothermal amplification (LAMP) has distinct advantages with regards to its simplicity, speed and the ease of performing without any need for sophisticated equipment. Over the last decade, LAMP has emerged as a potential tool for the detection of V. parahaemolyticus. Area covered: The literature search was restricted to LAMP assay and its variants for the detection of V. parahaemolyticus. The focus in this review is to enlist the various techniques that have been developed using the principle of the LAMP towards improved simplicity, sensitivity and specificity of the assay. Expert commentary: LAMP assay and its variants are significantly faster and require minimum accessories compared to other DNA based molecular techniques such as PCR and their types. Despite the availability of several versions, LAMP-based diagnostics is not the first choice for the detection of V. parahaemolyticus in the seafood sector. Our recommendation would be to explore the possibilities of developing cost-effective LAMP kits and implementing these kits as point-of-care diagnostic tools for rapid and sensitive detection of pathogenic V. parahaemolyticus.
Point of care
Point-of-Care Testing
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สารสนเทศทางทนตกรรม จยนศกษาความไวของเทคนคดเพลกซพซอารสำหรบการตรวจสอบ Vibrio parahaemolyticus ในกงขาวโดยใชยนเปาหมายสำหรบตรวจสอบ V. parahaemolyticus คอ tl (thermolabile hemolysin gene) และ tdh (thermostable direct hemolysin gene) พบวาความไวของเทคนคในการตรวจสอบแบคทเรยโดยตรงจากตวอยางกงขาว คอ 103 CFU/g แตการเพมปรมาณเชอกอนการทำพซอาร 6 ชวโมง จะชวยเพมประสทธภาพของการตรวจสอบไดดขน ดงนนจงมความเหมาะสมในการนำเทคนคดงกลาวมาใชในการตรวจสอบและเฝาระวงแบคทเรยนในตวอยางกงขาว เมอนำเทคนคดเพลกซพซอารมาตรวจสอบการปนเปอน V. parahaemolyticus ในกงขาวจำนวนทงหมด 60 ตวอยาง โดยสมเกบตวอยางจากจงหวดสมทรปราการชวงเดอนสงหาคม พ.ศ. 2557 ถงเดอนกนยายน พ.ศ. 2557 พบวามกงขาวจำนวน 42 ตว อยาง ทม ก ารปนเปอน V. parahaemolyticus (tl) คด เปน 70 เปอรเซน ต แตตรวจไมพบเชอ สายพนธกอโรคทมยน tdh การศกษานแสดงใหเหนวาเทคนคดเพลกซพซอารมประโยชนตอการนำไปใชเปนเครองมอเพอใหไดมาซงขอมลสำหรบนำไปใชในการเฝาระวงการแพรระบาดของเชอ และลดปญหาทางดานสาธารณสขทเกดจากแบคทเรยชนดน การออกแบบระบบ
Thermolabile
Hemolysin
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Vibrio parahaemolyticus has been considered as one of the most important food-borne bacterial pathogens. The loop-mediated isothermal amplification ( LAMP ) that amplifies DNA with high specificity and rapidity under isothermal conditions was applied for rapid detection of V. parahaemolyticus for the first time. A set of four primers, two outer and two inner primers, was designed specifically to recognize the thermolabile hemolysin gene ( tlh ) of V. parahaemolyticus. Genomic DNAs from 28 bacterial strains including 14 V. parahaemolyticus strains were amplified using LAMP, and no amplicon was observed in the other 14 bacterial strains. The detection limits of LAMP assay and pure cultures were 90 fg/LAMP mixture of V. parahaemolyticus genomic DNA and 24 cfu/mL, respectively. And for directly detecting V. parahaemolyticus in artificially contaminated food samples, the detection limit was 89 cfu/g. In addition, 40 seafood samples were tested by LAMP, and 8 of them were V. parahaemolyticus positive. Among the tested positive samples, 6 samples were also detected to be positive by conventional microbiological methods. These results suggest that detection of V. parahaemolyticus by LAMP is an effective and low-cost procedure with high specificity and sensitivity, and no specialized equipment required.
Thermolabile
Amplicon
genomic DNA
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Primer (cosmetics)
Hemolysin
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A hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus 06: K46 was purified by 55% ammonium sulfate fractionation and successive column chromatographies on DEAE-cellulose, hydroxyapatite, Sepharose 4B and Mono Q. The purified hemolysin was physicochemically and immunologically identical with the Vp-TRH (V. parahaemolyticus thermostable direct hemolysin related hemolysin) recently described in V. parahaemolyticus 03: K6 (Honda et al. Infect. Immun. 56: 961–965, 1988). This indicates that V. parahaemolyticus of Kanagawa-negative clinical isolates possessing not only 03: K6 but also different serotypes such as 06: K46 produce Vp-TRH. Production of Vp-TRH by most clinical isolates of Kanagawa-negative V. parahaemolyticus was also demonstrated. These results suggest the importance of Vp-TRH among clinical isolates of Kanagawa-negative V. parahaemolyticus.
Hemolysin
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Thermolabile
Hemolysin
Vibrio Infections
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Objective:To control contamination of Vibrio parahaemolyticus is often hampered by the lack of standardized methods and by the uncertainty associated with biochemical identification of the isolates.In this study,Vibrio parahaemolyticus was be examined thermolabile hemolysin(TLH) gene.Methods:PCR method was used to examine TLH gene isolates from clinical samples.Results:The TLH gene was found in all isolates.Conclusion:TLH gene has some value in detecting Vibrio parahaemolyticus.
Thermolabile
Hemolysin
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Thermolabile
Hemolysin
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Objective To develop an assay of loop-mediated isothermal amplification(LAMP) for detection and identification of Vibrio parahaemolyticus in basic laboratory.Methods The target of Vibrio parahaemolyticus was chosen from gyrB gene,four LAMP primers were designed and reaction conditions were optimized.Results: LAMP method was of high specificity for Vibrio parahaemolyticus.Through the amplification of 14 strains,Vibrio vulnificus was positive,and the other strains were negative.And the detection of LAMP method was finished within 60 minutes,and the detection limit was 25 cfu/ml.Totally 120 seafood samples were detected and positive rate was 62.5%.LAMP method was accord with traditional methods.Conclusion Comparing to traditional method,LAMP method is a good one:saving time,lower requirement of laboratory instruments and of good practicability for Vibrio parahaemolyticus detection.
Vibrio vulnificus
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