A Visual LAMP Assay for Detection of Chicken Gyrovirus 3
To establish a visual LAMP detection method for chicken gyrovirus 3(GyV3)and provide a detection method for rapid clinical diagnosis of GyV3 infection,a set of specific primers for loop-mediated isothermal amplification(LAMP)was designed according to the VP3 gene of GyV3.The amplification temperature,primer concentration,and reaction time of the visual LAMP diagnostic method were determined by optimization of the temperature,primer concentration,and reaction time.The specificity,sensitivity,stability and reliability of the chicken GyV3 LAMP method were verified through the specificity test,sensitivity test,repeatability test and detection of clinical samples.The reactions were optimized in a final volumes of 20.0 μL,including 1.0 μL DNA template,10.0 μL 2×Master Mix,2 μL mixture of inner primers GyV3-FIP and GyV3-BIP(working concentration 16.0 μmol/L),2.0 μL mixture of outer primers GyV3-F3 and GyV3-B3(working concentration 2.0 μmol/L),2.0 μL mixture of loop primers GyV3-LF and GyV3-LB(working concentration 8.0 μmol/L),3 μL ddH2O.The amplification procedures of GyV3 LAMP were determined:reaction at 66℃for 20 min,followed by heating at 80℃for 5 min.The visual LAMP assay for the detection of chicken GyV3 could specifically detect GyV3;the lowest limit of GyV3 detection was 101 copies/μL;the coefficients of variation of intra-batch and inter-batch assay between groups were both less than 5%;the coincidence rate between the detection results and the conventional PCR detection results was 100%.The visual LAMP assay for the detection of chicken GyV3 can specifically detect GyV3,with the advantages of good specificity,high sensitivity,and low pollution.The detection results can be judged by naked eye observation,which is suitable for rapid clinical screening of GyV3.
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