Determination of ABO blood group genotypes using the real-time loop-mediated isothermal amplification method

Background Enterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP. Methods A rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples. Results Positive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP. Discussion In this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.

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Development of a real time loop-mediated isothermal amplification method for detection of Senecavirus A

Journal of Virological Methods ◽

10.1016/j.jviromet.2018.08.005 ◽

2018 ◽

Vol 261 ◽

pp. 98-103 ◽

Cited By ~ 7

Author(s):

Fanwen Zeng ◽

Feng Cong ◽

Xiangnan Liu ◽

Yuexiao Lian ◽

Miaoli Wu ◽

...

Keyword(s):

Real Time ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification ◽

Amplification Method ◽

Time Loop

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Rapid Identification of Pseudomonas fluorescens Harboring Thermostable Alkaline Protease by Real-time Loop-mediated Isothermal Amplification

Journal of Food Protection ◽

10.4315/jfp-21-272 ◽

2021 ◽

Author(s):

Lianxia Hu ◽

Shufei Zhang ◽

Yuling Xue ◽

Junhua Han ◽

Huaxi Yi ◽

...

Keyword(s):

Phylogenetic Tree ◽

Pseudomonas Fluorescens ◽

Real Time ◽

Alkaline Protease ◽

Raw Milk ◽

Rapid Identification ◽

Isothermal Amplification ◽

The Real ◽

Loop Mediated Isothermal Amplification ◽

Time Loop

Thermostable alkaline protease (TAP) harbored by Pseudomonas fluorescens decomposes protein in milk and dairy products, leading to milk and dairy product spoilage during storage. Thus, a specific, sensitive, rapid, and simple method is required to detect TAP-harboring P. fluorescens. Two sets of primers targeting the aprX and gyrB genes of P. fluorescens were designed. The detection system and conditions were optimized, and a real-time loop mediated isothermal amplification (real-time LAMP) method was developed for the simultaneous detection of TAP-harboring P. fluorescens in two separate reaction tubes. The phylogenetic tree targeting aprX showed that P. fluorescens and P. lurida clustered on the same branch. The phylogenetic tree targeting gyrB showed that P. fluorescens clustered on the same branch with 95% confidence value, whereas P. lurida clustered on different branches. DNA of 16 strains of P. fluorescens and 34 strains of non-P. fluorescens was detected by real-time LAMP. TAP-harboring P. fluorescens can only be identified when the real-time LAMP detection results of both aprX and gyrB are positive. The dissociation temperatures of aprX and gyrB in the real-time LAMP amplified products were approximately 90.0 °C and 88.0 °C, respectively. The detection limits of the real-time LAMP targeting aprX and gyrB were 4.9 CFU/reaction in pure culture and 2.2 CFU/reaction in skimmed milk. The coefficient of variation of the repeatability test was less than 2%, indicating that the established real-time LAMP of P. fluorescens targeting gyrB and aprX has good stability and repeatability. Two-hundred raw milk samples were tested for the presence of TAP-harboring P. fluorescens by real-time LAMP in 3 h, and the coincidence rate of the results with those obtained using the traditional method, which takes at least 5-7 d, was 100%. Real-time LAMP will be a practical and effective method for accurate and rapid identification of TAP-harboring P. fluorescens in raw milk.

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