Establishment And Application Of Zipper Probe Loop-Mediated Isothermal Amplification Technology To Extract Rotavirus Free Nucleic Acid

Objective It aims to develop a rapid and simple rotavirus nucleic acid test without nucleic acid extraction reagent for rotavirus detection,combine it with reverse transcription loopmediated isothermal amplification（RT-LAMP）system.and establish a rapid,sensitive and specific nucleic acid-free probe testing method,RT-LAMP testing method,for real-time and rapid detection of rotavirus（RoV）in feces.Methods①Three nucleic acid-free reagent fractions were formed by orthogonal tests to extract hepatitis B virus DNA from blood specimens and Mycoplasma solium RNA from urine specimens,and the effect of nucleic acid extraction was tested by PCR to verify the sensitivity,reproducibility and accuracy of this nucleic acid-free extraction reagent.②Rotavirus-specific LAMP primers and zipper probes were designed,the screening of three pairs of LAMP primer ratio,probe ratio,enzyme amount,Mg2+ concentration and template amount using orthogonal design was optimized,rotavirus real-time fluorescent probe method RT-LAMP system was established,and its specificity,sensitivity and reproducibility were evaluated.③The abovementioned nucleic acid-free extraction reagents were combined with real-time fluorescent probe RT-LAMP system to establish a nucleic acid-free extraction probe RT-LAMP method for rotavirus detection,to verify its reproducibility and specificity,and to compare the positive rate of common PCR and nucleic acid-free extraction probe RT-LAMP detection methods with clinical specimens.Results ①Only one of the three nucleic acid-free extraction reagents could successfully extract nucleic acids,with the following components:1.5%of Tween20,0.5%of Triton 100,and 1.5%of rhamnolipid.And the optimal pH is 9.The ratio of nucleic acid-free reagent to sample spiked amount was 6:1,and the extraction time was 10 minutes.②The lower limit of detection of rotavirus probe method RT-LAMP method was 10 Copies/μl,no non-specific amplification occurred,and the coefficient of variation of the mean Ct value of the amplification curve of the reproducibility experiment were less than 5%.③50 rotavirus specimens tested by nucleic acid-free extraction-based probes were consistent with the results of common PCR testing result and were more sensitive than clinical rotavirus antigen assays.Conclusion ①The nucleic acid-free extraction method established in this study can extract RNA or DNA from different types of samples such as urine,blood and feces,which takes less time and has high extraction rate,and the extracted nucleic acids meet the requirements of PCR,LAMP and other molecular biology detection methods.②A probe RT-LAMP method was developed in this study to detect rotavirus in real time,which is simple,rapid,sensitive,specific and reproducible.③The nucleic acid-free extraction-based probe RT-LAMP method was developed as a rapid,specific,sensitive and reliable method for the detection of rotavirus mentioned in this study.