Comparative rapid identification of Salmo salar, Oncorhynchus mykiss, and Oncorhynchus keta components based on loop-mediated isothermal amplification and quantitative polymerase chain reaction

Background: Sensitive and definitive diagnostic tests are required for timely treatment of leprosy and to control its transmission. Aim: In the present study, we report the development of loop-mediated isothermal amplification assay using six primers targeting the RLEP gene sequence uniquely present in Mycobacterium leprae. Methods: Tissue punch samples (n = 50) and slit aspirates (n = 50) from confirmed cases of leprosy (M. leprae positive by quantitative polymerase chain reaction), reporting at the Department of Dermatology, Safdarjung Hospital, New Delhi, were analyzed using newly developed closed tube loop-mediated isothermal amplification assay. The sensitivity and specificity; positive predictive value, negative predictive value and accuracy were calculated using MedCalc statistical software. Results: The loop-mediated isothermal amplification assay specifically amplified M. leprae genomic DNA with an analytical sensitivity of 100 fg. About 47 Out of the 50 quantitative polymerase chain reactions confirmed M. leprae positive tissue samples, 47 were positive by loop-mediated isothermal amplification assay (sensitivity 94%; 95% confidence interval 83.5%–98.8%) while only 31/50 were positive by histopathology (sensitivity 62%; 95% confidence interval 47.2%–75.4%). Using slit aspirate samples of these 50 patients, 42 were positive by both quantitative polymerase chain reaction and loop-mediated isothermal amplification assay (sensitivity 84%; 95% confidence interval 70.9%–92.8%) while only 23/50 (sensitivity 46%; 95% confidence interval 31.8%–60.7%) were positive by microscopy. Limitations: In the present study, the leprosy patient cohort was not uniform, as it comprised of a lower number of paucibacillary cases (22%) compared to multibacillary (78%) cases. Conclusion: Loop-mediated isothermal amplification assay established here provides a rapid and accurate diagnostic test for leprosy in terms of sensitivity and specificity. The assay is simple to perform in comparison with other molecular techniques (polymerase chain reaction/quantitative polymerase chain reaction) and has potential for field applicability.

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Species identification of felis and vulpes by a novel loop-mediated isothermal amplification assay in fur products

Journal of Engineered Fibers and Fabrics ◽

10.1177/1558925018820720 ◽

2019 ◽

Vol 14 ◽

pp. 155892501882072

Author(s):

Shunji Yu ◽

Wenjia Gu ◽

Yi Yu ◽

Qinfeng Qu ◽

Yi’nan Zhang

Keyword(s):

Polymerase Chain Reaction ◽

Species Identification ◽

Quantitative Polymerase Chain Reaction ◽

Cost Effective ◽

Isothermal Amplification ◽

Chain Reaction ◽

Species Identity ◽

Loop Mediated Isothermal Amplification ◽

Amplification Method ◽

Polymerase Chain

In the chaotic market of fur goods, genetic distinction is increasingly important for identifying species. A vast diversity of species identification methods has been proposed, while little is developed, particularly those easy, fast, and cost-effective ones. In this study, a simple and reliable novel loop-mediated isothermal amplification method for identifying cytochrome c oxidase I of felis and vulpes was established. It saves laborious post–polymerase chain reaction procedures and shortens the time for high-fidelity gene amplification. The sensitivity of this method for felis and vulpes identification, which is well matched to quantitative polymerase chain reaction, could be 10 or 1.0 pg, respectively. Predominantly, the sensitivity of loop-mediated isothermal amplification is more tolerant to those polymerase chain reaction inhibitors such as pigments, dyes, or other fur ingredients, compared to quantitative polymerase chain reaction. Even without costly specialized equipment, a water bath is sufficient for genetic distinction. Our approach is a new technique with broad application perspective, such as on-site species identity tests, commercial fraud, and wildlife crimes.

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