Monkey Parainfluenza Virus Detection Method And Its Preliminary Application,

Simian parainfluenza virus 5 (SV5) is a type of RNA virus which is an ubiquitous virus in simian's, and also is a frequent contamination from simian's cell cultures. To guarantee the quality of laboratory animal productions, and the quality and safety of biological products from simians for human use, it is necessary to detect SV5 in the process of quality control of laboratory animals and biological products. The purpose of our research work is to establish the methods based on molecular biological and serological technologies to detect SV5 in related biological products.To establish the PCR method for the detection of SV5, according the primer designing principle, a pair of primers were designed to aim directly at the SH gene (292bp), which is the most conservative gene fragment in the SV5 genome. Viral RNA was extracted from the Vero cell culture infected with SV5, and cDNA was reverse-transcripted by using Random Primer as 3' out primer. An approximately 237bp idio-fragment was amplified from the cDNA by the designed primers. The amplified fragment was sequenced and proved to be the SH gene sequence of SV5 genome. The conditions of the RT-PCR were then optimized by a series of gradient experiments. Sensitivity of the method was determined as the minimum RNA concentration of 2.1ng/μl. For initial application of the method, samples including simian organ from Beijing Research Institute of Biological Products, ordinary hamster's organ from six domestic research institute of biological products, SPF hamster's organ from Beijing Lab Animal Center, biological products from simian (OPV), and five commonly used passage cells from simian kidney, were tested by the new method for the SV5 nucleotide gene.Results came out that no SV5 nucleotide gene were detected in above specimens.To detect the distribution conditions of SV5 antibody in laboratorial simians, two antibody detection methods, IFA and IEA, were established using the simian SV5-positive serum provided by Lab Animal Research Institute of China Academy of Medical Science. SV5 antigen was prepared by cell culture method, and the hamster's SV5 positive serum was prepared by immunizing hamsters. Then the sensitivity of the two methods was determined. The two new methods were initially applied on simian serum samples and hamster serum samples from different sources. Result came out that 89 of 335 sepcimens positively reacted in total 614 simian serum soecimens,and no positive reaction in total 119 hamasters' serum specimens.