Development and comparative evaluation of loop mediated isothermal amplification (LAMP) assay for simple visual detection of orf virus in sheep and goats

2015 ◽  
Vol 29 (3) ◽  
pp. 193-195 ◽  
Author(s):  
G. Venkatesan ◽  
V. Bhanuprakash ◽  
V. Balamurugan
Author(s):  
Can Wang ◽  
Ziheng Xu ◽  
Xuejiao Hou ◽  
Min Wang ◽  
Chenyu Zhou ◽  
...  

Salmonella is one of the major pathogenic bacteria causing food-borne diseases. The rapid detection of Salmonella in food is of great significance to food safety. In this study, the loop-mediated isothermal amplification (LAMP) method was developed and the primers were designed targeting the invA gene of Salmonella. Then, the standard samples of recombinant invA-plasmid and 100 retail meat samples were tested by LAMP and compared with the results tested by the conventional PCR and the routine China National Food Safety Standard Methods for Food Microbiological Examination-Salmonella (GB/T4789.4-2016), respectively. The results showed that Salmonella strains of 8 different serotypes were amplified successfully by the developed LAMP assay. And, it was 1000-fold more sensitive than the conventional PCR with the analytical sensitivity of 8×102 copies/μL of the standard sample of invA-plasmid. The results were visualized directly by adding Calcein/MnCl2 into the LAMP reaction tube and the positively amplified products turned green after an incubation of 2 min. In the parallel detection, the positive rate of Salmonella by the LAMP assay was highly correlated with the routine China national standard method. The results of the study demonstrated that the developed LAMP assay is a simple, rapid, strongly specific, highly sensitive and visual detection method for Salmonella.


2021 ◽  
Vol 14 (2) ◽  
Author(s):  
Azi Simon Simon ◽  
Suresh Kumar Subbiah ◽  
Leslie Thian Lung Than ◽  
Malina Osman ◽  
Rukman Awang Hamat

Background: Group A Streptococcus (GAS) causes a wide array of clinical manifestations ranging from mild pharyngitis to suppurative and non-suppurative severe debilitating diseases. Hence, a simple, rapid detection method with high sensitivity and specificity is needed. Objectives: This study embarked on the visual detection of the streptococcal pyrogenic exotoxin B (speB) gene by Real-time Turbidimetry and Loop-mediated Isothermal Amplification (RT-LAMP) methods. The real-time monitoring of the sigmoidal graph generated from a turbidometry method was incorporated in the assay. Methods: The amplification of the speB gene was virtually observed in real-time monitoring of the graph (sigmoidal curve) generated via a turbidimeter, thus providing a “guide” to accurately estimate the time to positivity for the gene detection. Results: The targeted gene was detected at 15 min but was optimally amplified within 45 min at an isothermal temperature of 63°C with 100% specificity using an established set of primers. The formation of sigmoidal curves was correlated with other visual observations by the naked eye (from orange to green), ultra-violet light (green fluorescence), and agarose gel electrophoresis. The improved detection limit of the real-time RT-LAMP assay was also observed compared to conventional PCR assay (0.001 pg/µL versus 1 ng/µL). Conclusions: In conclusion, this improved visual detection of RT-LAMP assay could provide additional insight for rapid, cost-effective, and reliable identification of GAS via speB gene detection in low or middle-income countries. It could also be a very important tool to improve the healthcare management of patients infected with GAS in the future.


2016 ◽  
Vol 56 (2) ◽  
pp. 142-147 ◽  
Author(s):  
Bikash R. Prusty ◽  
Pallab Chaudhuri ◽  
V. K. Chaturvedi ◽  
Mohini Saini ◽  
B. P. Mishra ◽  
...  

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