Dataset Information

Rapid detection of infectious bursal disease by loop-mediated isothermal amplification for field analysis.

ABSTRACT: Infectious bursal disease (IBD) is an immunosuppressive, acute and highly contagious illness of growing-poultry stock infected with infectious bursal disease virus (IBDV). It is common in Pakistan, causing potential economic losses throughout the year. The objective of the study is to propose a rapid, sensitive and specific diagnostic tool, and compare it with existing commonly used reverse transcriptase polymerase chain reaction (RT-PCR) method for IBDV. Different primers were used for RT-PCR and reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) to target the IBD virus. RT-LAMP primers showed prodigious specificity without cross reaction to the other animal pathogens. Moreover, RT-LAMP was found to have 10 times higher selectivity for IBDV identification as compared to RT-PCR. RT-LAMP detected 9.2% more field samples than RT-PCR. Sequences of PCR products were determined and phylogenetic analysis of research isolates revealed its maximum similarity with indigenous and Indian IBDV isolates. RT-LAMP was found to be simple, specific, less laborious and a better technique as compared to RT-PCR for quick analysis. In general, RT-LAMP was declared positive on observing turbidity or adding fluorescence staining reagent such as SYBR Green I. The options of direct use of field sample homogenate and viewing directly the peaks in the graph shown on a monitor/laptop have made it much more convenient and time saving than gel based RT-PCR.

SUBMITTER: Khan RSA

PROVIDER: S-EPMC6056140 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Rapid detection of infectious bursal disease by loop-mediated isothermal amplification for field analysis.

Khan R S A RSA Ali W W Kiran S S Shah M S D MSD Tahir Z A ZA Habib M M

Iranian journal of veterinary research 20180101 2

Infectious bursal disease (IBD) is an immunosuppressive, acute and highly contagious illness of growing-poultry stock infected with infectious bursal disease virus (IBDV). It is common in Pakistan, causing potential economic losses throughout the year. The objective of the study is to propose a rapid, sensitive and specific diagnostic tool, and compare it with existing commonly used reverse transcriptase polymerase chain reaction (RT-PCR) method for IBDV. Different primers were used for RT-PCR a ...[more]

PMID: 30046320

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Project description: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.

Dataset Information

Rapid detection of infectious bursal disease by loop-mediated isothermal amplification for field analysis.

Publications

Rapid detection of infectious bursal disease by loop-mediated isothermal amplification for field analysis.

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