RT-PCR For Clinical Detection And Gene Sequence Analysis Of Rubella And Measle

Rubella caused by rubella virus(RV) is a mild, limited and infective illness that presents with fever and rash. It spreads by respiratory passage and harm the health of adolescence. When rubella infection is acquired in the early 3 months of pregnancy,it can result in fetal death or in the birth of an infant with congenital rubella syndrome(CRS). Measle is a infective disease by measles virus(MV) that cause the child to develop fever, catarrhus and allover body maculopapule. The typical cases are scarce,so the identification between RV and MV is difficult.Current laboratory diagnostics is mainly based on cell culture, serology and anti-rubella/measle IgM could be detected after onset. But these methods can appear false-positive or false-negative disturbed by time, antibody interference,etc. Otherwise, they can't express the mutation of genes. RT-PCR is a sentive and rapid assay, which can be used in conjunction with serology testing for diagnosis of rubella and measles infection. In this study we will analyze the characteristics and the viariations of RV/MV genes circulated in DaLian. At the same time we will obtain the best type of the clinical specimens.Materials and Methods: 1.Blood, urine and oral specimens collected from 59 clinical doubtful patients in DaLian are tested in this study.Total RNA extraction and reverse transcription nested polymerase chain reaction (RT-PCR) by two pairs of RV/MV specific primers. From the sizes of the RT-PCR products running on 1% agarose gels decide the positive/negative. 2.The positive amplifications are purifyed and further sequenced. 3.All specimens used in this study were tested for RV/MV specific IgM. 4.Phylogenetic trees are made on the basis of positive amplifications nucleotide sequences and other areas sequences in the Genebank.Results: 1.Three specimens(5.08%) produced a PCR amplicon of 592bp in size which is same with RV positive control are the RV positive. Two specimens(3.39%) produced a PCR amplicon of 547bp in size which is same with MV positive control are the MV positive. 2.The percent divergences of the RV positive specimens respectively are 41~# 2.74%, 37~# 2.19%, 43~# 2.19%. The percent divergences of the MV positive specimens respectively are 46~# 9.39%,36~# 7.27%. 3.Five instances are RV IgM positive.Eight instances are MV IgM positive.Three instances are MV IgM suspect positive. 4.Two blood specimens,three oral fluid and two culturing cells are RV&MV RT-PCR positive. Tree blood speciments, three urine speciments and six oral fluids are RV&MV-ELISA positive. 5.Two MV RT-PCR positive instances correspond with MV IgM positive. Two RV RT-PCR positive correspond to MV IgM positive. One is RV RT-PCR positive and no sorrespongding antibody. Five are RV IgM positive and no antigen found. Seven are MV IgM positive and no antigen found. 6.The sequence analysis of the E1 coding gene of the three RV isolates similar with genotypeⅠrepresentative strain RA27 at 96.9%～97.5% and with genotypeⅡrepresentative strain BRDⅡat 91.8%～92.5%. Among them SXM-37~#RV and SXM-43~#RV are the same, similar with JUDITH at 99.2%. SXM-41~#RV similar with RA27-3 at 99.2%. While SXM-44~#MV is similar with DL6China97n at 99.3%. SXM-46~#MV is similar with hebei05-18 at 99.1%.Conclusions: 1.We should do RT-PCR detect during the two or three days after onset.If the product is positive,we can reach the final diagnosis. If the product is negative, then we should test the antibody during two to fifteen days.Because RV&MV IgM has cross reaction, we must distinguish them by RT-PCR. 2.This study demonstrates that the E1 gene of RV and the N gene of MV are steady and can be used for genotyping and epidemiological surveillance. 3.Blood and oral fluid are the better for RT-PCR test.Blood, urine and oral fluid are all can be used for ELISA.But blood is prior to oral fluid,oral fluid is prior to urine. 4.Phylogenetic analysis of clinical samples investigated in this research and comparison with other available sequences in GenBank demonstrates that the three RV instances fall into GenotypeⅠ. Two instances coming from one virus strain are similar with the positive control. The other one is similar with the standard vaccine strain. 5.Thephylogenetic and sequence analysis of the N gene of the two MV instances indicate that all are members of genotype H1. One is similar with the DaLian native strain. Another is similar with HeBei strain, which maybe the immigrated strain.