Research of fluorescent reagent in detection of IBV by loop-mediated isothermal amplification(LAMP)

【Objective】 In order to provide support for infectious bronchitis virus detection,a novel method namely loop-mediated isothermal amplification(LAMP)was reported using fluorescent reagent as the product judgment.【Method】 With one set of primers containing six special primers targeting eight part of 5a+5b gene obtained from NCBI,the LAMP system was optimized by sifting the time,temperature and concentration of all components.Then specificity and sensitivity test was conducted.SYBR Green I and calcein/Mn2+ reagent were respectively used in LAMP product judgment with naked eyes.In order to compare the LAMP and PCR method,sixty clinical samples were detected.【Result】 The LAMP method was successfully established,and the reaction could complete within 1 hour at 63 ℃ with favorable sensitivity and specificity.The detection limit of the assay was 1 copy virus,which was 100 times more sensitive than traditional PCR.In the fluorescent reagent test,SYBR Green I and calcein reagent which contained 0.05 mmol/L calcein and 0.6 mmol/L Mn2+ were respectively used in LAMP product judgment,and the result showed strong colorimetric comparison between positive and negative and was consistent with agarose gel electrophoresis.In the detection of 60 clinical samples,49 and 42 samples were respectively positive by LAMP and PCR assays.【Conclusion】 LAMP assay with fluorescent reagent provided a useful tool for infectious bronchitis virus rapidly detection.