scholarly journals A Complementary Isothermal Amplification Method to the U.S. EPA Quantitative Polymerase Chain Reaction Approach for the Detection of Enterococci in Environmental Waters

2017 ◽  
Vol 51 (12) ◽  
pp. 7028-7035 ◽  
Author(s):  
Claudia Kolm ◽  
Roland Martzy ◽  
Kurt Brunner ◽  
Robert L. Mach ◽  
Rudolf Krska ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Environmental Waters ◽  
Polymerase Chain Reaction Approach ◽  
Amplification Method ◽  
Polymerase Chain ◽  
The U.S

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

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.

scholarly journals Development of a novel loop-mediated isothermal amplification assay for rapid detection of Mycobacterium leprae in clinical samples

2021 ◽  
Vol 0 ◽  
pp. 1-7
Author(s):  
Shweta Joshi ◽  
Vanila Sharma ◽  
V. Ramesh ◽  
Ruchi Singh ◽  
Poonam Salotra
Keyword(s):  
Polymerase Chain Reaction ◽  
Confidence Interval ◽  
Quantitative Polymerase Chain Reaction ◽  
Mycobacterium Leprae ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Assay Sensitivity ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
Polymerase Chain

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.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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