Establishment Of The Method Of A Triple Fluorescence RT-LAMP Without Nucleic Acid Extraction And Development Of A Kit For Detection Of Feline Calicivirus、feline Parvovirus、feline Herpesvirus-1

Feline calicivirus(FCV),Feline panleukopenia virus(FPV)and Feline herpesvirus-1(FHV-1)are three major infectious viruses that seriously damage the health of felines,which are highly contagious and have a high mortality.The commonly used laboratory detection methods mainly included electron microscopy observation,PCR,real-time quantitative PCR(q PCR),recombinase amplification(RPA),enzyme linked immunosorbent assay(ELISA),colloidal gold immunochromatography and so on.However,it was difficult to be widely used in clinical detection due to high requirements on experimental equipment and environment,time-consuming,high cost,high false positives or other shortcomings.Therefore,it was very important to establish a quick and simple detection method for FCV,FPV and FHV-1 for the diagnosis and prevention of virus infection.Loop-mediated isothermal amplification(LAMP)was a rapid nucleic acid isothermal amplification technique that could amplify DNA fragments under constant temperature.It was simple,high sensitive and specific,and played an important role in the detection of bacteria,fungi,viruses,parasites,sex identification and so on.In addition,LAMP could directly use complex samples without nucleic acid extraction,which could reduce the cost and time of analysis.In recent years,the multiple LAMP technology,which detected multiple pathogens simultaneously by designing specific primers for different target genes,had also become a hot spot at home and abroad.Therefore,the study of detection of common viruses based on LMAP was of great significance for clinical disease identification of small animals and clinical application of LAMP technology.This study was aimed to use LAMP technology to establish a triple RT-LAMP with high sensitivity,high specificity and good reproducibility,which directly used the clinical sample without nucleic acid extraction to simultaneously detect FCV,FPV and FHV-1,and develop a rapid detection kit.Experiment Ⅰ.The establishment of fluorescence(RT)-LAMP detection methods without nucleic acid extractionIn this study,(RT)-LAMP specific primers and assimilation probes were designed based on the conserved sequences of FCV,FPV and FHV-1 genes.The 5 ’end of each loop primer LF was labeled with different fluorescent groups and observed under different fluorescence channels.Fluorescence(RT)-LAMP detection methods for FCV,FPV and FHV-1 were established,and their specificity and sensitivity were investigated.The FCV,FPV and FHV-1 nucleic acid templates were detected by(RT)-LAMP,respectively,and compared with the direct fluorescence(RT)-LAMP and(RT)-PCR results of the virus stock solution.The results showed that there was no cross reaction among FCV,FPV,FHV-1,feline coronavirus(FCo V)and Mycoplasma by Fluorescence(RT)-LAMP detection methods without nucleic acid extraction.The lowest limits of detection for FCV,FPV and FHV-1 were 45.7 fg/μL,5.57 fg/μL and 3.72 fg/μL,respectively.they were 10 times higher than those of(RT)-PCR.The established fluorescence(RT)-LAMP detection methods without nucleic acid extraction had strong specificity and high sensitivity,which provided supports for the clinical detection of FCV,FPV and FHV-1.Experiment Ⅱ.The establishment of the nucleic acid extraction-free triple fluorescence RT-LAMP detection method and the development of the kitThis part of test was aimed to establish a triple fluorescent RT-LAMP detection method without nucleic acid extraction which could simultaneously detect FCV,FPV and FHV-1.The combination of different primer concentrations were optimized to determine the best primer ratio.The RT-LAMP conditions were optimized to develop a nucleic acid extraction-free triple fluorescence RT-LAMP detection kit,and its specificity,sensitivity and stability were investigated.The results showed that the fluorescence RT-LAMP primer concentration of FPV and FHV-1 was reduced to 1/4 of the original concentration,and the fluorescence RT-LAMP primer concentration of FCV was maintained.The method could detect FCV,FPV and FHV-1 at the same time,and has no cross reaction with FCo V and Mycoplasma.The lowest limits of detection for FCV,FPV and FHV-1 were 45.7 fg/μL,5.57 fg/μL and 3.72 fg/μL,respectively.It was consistent with the results of nucleic acid extraction-free fluorescence(RT)-LAMP.The results of the developed kit were consistent with clinical diagnostic kit,PCR or colloidal gold immunoassay strip.The coincidence rate was 92.5%.The established nucleic acid extraction-free triple fluorescence RT-LAMP detection method in this study and the developed detection kit had strong specificity,high sensitivity and good stability.The whole detection process could be completed within 1hour by using only the clinical sample without nucleic acid extraction,which was an effective method for rapid clinical detection of FCV,FPV and FHV-1.