Establishment And Preliminary Application Of Real-time Fluorescence Quantitative PCR For Detecting The Giant Panda Rotavirus

The giant panda(Ailuropoda melanoleuca)is an endangered wildlife species endemic in China.Under the fragmentation of large-scale habitats,and the low naturally breeding rates of the isolated small population,the off site conservations against the captive giant panda is particularly important.The infection of Giant Panda Rotavirus(GPRV)often causes diarrhea and mortality of panda cubs,which is a disease that seriously harms the giant panda's off site conservations.Due to the diverse diagnostic methods of rotavirus that each has its own advantages and disadvantages,for the giant panda diarrhea disease,quickly and accurately distinguish whether caused by rotavirus infection,provide direction for clinical treatment of win precious time and guidance.In recent years,real-time fluorescence quantitative PCR(qPCR)technology has played a major role in the diagnosis of public health epidemics and is considered to be an extremely efficient diagnostic method.This thesis,which bases on the GPRV VP4 gene sequence that available from GenBank database,tests 1-pair-primer-PCR to establish a rapid and accurate quantitative qPCR detection method for GPRV.And by optimizing the primer concentration,annealing temperature and cycle number,a standard curve is established,so that the sensitivity of this method is 1.0×10~0copies/?L;simultaneously by detected the recombinant plasmid nucleic acid sample at different dilutions,which the sensitivity is 1.0×10~2copies/?L.Therefore,the sensitivity of GPRV qPCR is at least 100 times higher than that of conventional RT-PCR.The results were negative while the nucleic acids of CDV,CPV,CAV-1 and CAV-2were detected by GPRV qPCR,it shows that this method had a high specificity and reliable results.The repeatability test showed that the coefficient of variation(CV)between batches and within batches was less than 1%,indicating that this method had a good repeatability and stable results.Therefore,this GPRV qPCR can meet the need of a rapid,accurate and sensitive diagnosis of infection in giant panda rotavirus diarrhea samples.This GPRV qPCR was used to detect 227 panda feces from different periods and individuals.The detection rate of conventional RT-PCR of samples in Base A was 8.1%in June 2015 while the detection rate of qPCR was 13.5%,and the detection rate of RT-PCR of samples in Base B was 14.3%during 2011 to 2013 while the detection rate of qPCR was 19%.Also,The detection rate of RT-PCR of samples in Base B was 0%in June 2015 while the detection rate of qPCR was 12%.In each detection,the qPCR showed a better sensitivity.At the same time,in the research and application,it is found that it has a great guiding role in the background investigation and disease prevention and control of pandas carrying rotavirus.Therefore,the GPRV qPCR method established in this thesis can better meet the needs of epidemic background investigation of the giant panda rotavirus.It is of great value to benefit the health management of giant panda population,optimize the external display of giant panda,guide the wild released giant panda selection,and improve the disease surveillance of wild giant panda.