Development And Application Of Rabies Virus Real Time Fluorogenetic Quantitative RT-PCR Detection Kit

Rabies is a deadly enzootic disease caused by rabies virus that attacks the central nervous system and causes acute encephalitis.It is reported that worldwide about 40,000-70,000 people die from rabies each year and most of them occurred in the developing country,of which approximately 98% occur in Asia. In China, which now has the second highest rates of illness and death from human rabies cases in the world after India.The reprots of epidemic diseases issued by China CDC showed that the human death toll is more than 2000 every year and has been increasing since 2002; in the year 2007 alone total case numbers were 3302 and a peak of the current epidemic is yet to be reached. These facts indicate that rabies, the important enzootic disease, bears big threat for the public health.Classical rabies virus, belonging to the Lyssavirus genus within the rhabdoviridae family, and its genome consists of a non-segmented, single-stranded negative-sense RNA of about 11928-11932 nt, comprising five genes that encode the nucleoprotein (N), phosphoprotein (P),matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (large protein, L).The N gene.because of its most conserved among the Lyssaviruses and high copies in replication of virus.is a ideal target for genotyping and diagnosis.Sequence comparison of the N gene, has provided evidence for division of the Lyssavirus genus into seven established genotypes and other four additional genotypes. Based on the phylogentic analysis of the current rabies virus strains in China, it showed that all RVs in our country belong to Lyssavirus genotype 1 and other genotypes has not been described.It is currently recommended by the WHO Expert Committee that the fluorescent antibody test is the gold criteria for diagnosis of rabies,with the mouse inoculation test (MIT) being used as confirmatory backup procedures and a method to isolate virus, and the detection of viral nucleic acids by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used for diagnosis and genotyping and, in combination with nucleotide sequencing, for epidemiological investigations.Because of intuitionistic detection result and being easily identified, These techniques have been applied widely in detecting rabies.However,FAT is not fit for detection of decomposing samples and liquid samples such as salivary glands and cerebrospinal fluid (CSF).And MIT needs to take a long experiment period and sacrifice animal welfare. The disadvantages of RT-PCR are the potential for cross-contamination and test failing. In conclusion,these methods are not recommended to detect the massive clinical samples. Nevertheless,the real time PCR offers a sensitive,specific,rapid and high-throughput assay for virus detection.And with the development of this technology at home and abroad, the real time PCR on rabies virus showed good application prospect.Given the wide variety of rabies susceptible animal species and diverse characteristics of distribution of the current rabies virus strains in our country, one Pair of specific Taqman probe and primers are designed according to integrated arrangement and comparison of partial N gene squences of 31 RV downloaded from the Genbank. And then a one-step real-time RT-PCR(qRT-PCR) was established to detect the rabies virus,through the optimizaion of reaction system and conditions. On this basis, the technical work-flow of detection kit was designed,and eventually Rabies virus Real Time Fluorogenetic Quantitative RT-PCR Detection kit was successfully developed. This test method can detect RV specifically.especially the rabies epidemic strains in China.According to the analysis of BLAST between the probe/primers and corresponding sequences of RV downloaded from the Genbank,only a few sequences mismatches exists,However,mismatches on primer and/or probe binding sites did not affect amplification or detection.The specificity assay showed that the method could only detect the RV, not other six lyssaviruses from genotype 2 to 7, and other viruses such as CDV,CPV,CAV,VSV,JEV.The stability assay showed that the Ct value of five repeated samples are less than 5% of variation coefficient, which proves this method to be a good stability in detection of Intracellular and brain tissue rabies virus.3 vaccine strains and 7 street strains from different clusters/clades and different areas were tested positive by qRT-PCR,which indicated that the method was able to detect a wide range of rabies virus strains in our country.For determination of the relative sensitivity of the qRT-PCR assay compared to nested RT-PCR,a 10-fold serial dilution of total RNA extracted from BHK-21 infected with rabies virus SRV9 was prepared in nuclease-free water and used as a standard samples to quatify viral titers in the clinical samples.The sensitivity assay showed that The detection limit of the established method was 4.68 TCID50, which is equally sensitive with our lab-developed nested RT-PCR.And there is a good line relationship between Ct value and the start density of RNA template, with the correlation coefficent r 0.999, slope-3.412. standard equation: Ct=-3.495 log 10TCID50+33.29.To validate the method 29 fresh and 5 decomposed dog brain specimens submitted to our laboratory for rabies confirmation were detected in comparison with OIE golden diagnostic standard methods FAT and MIT and routinely-used nested RT-PCR. In the result, the qRT-PCR was equally sensitive with nested RT-PCR and more sensitive than FAT and MIT,which is particularly useful for decomposed brain tissue.According to the production process of the reagent kit,the reproducibility trial and stability trial for 12 monthes duration have synthesized to evaluate several indexes of the reagent kit that has been built up using three batches of qualified qRT-PCR reagent,three batches are 2009001,2009002, and 2009003.In order to apply the developed kit to monitor production quality of the rabies virus vaccines and study the cellular adaptability of street strains, viral loads was quantified by this kit in Intracellular rabies virus (SRV9,hao,WS,FJ02)from F1 to F8,in comparison with 50% tissue culture infective dose (TCID50) determination test.Comparison showed that the result discrepancy between the two method within 1OTCID50/100μl,indicating that the kit could replace the TCID50 determination method to monitor vaccines production quality and study the virus cellular adaptability.In this study,we used the developed kit for the rabies virus detection from incubated decomposed brain tissues, and the results showed that the kit and nested RT-PCR cound detect the virus nucleic acid after 17d,which were more sensitive than FAT and MIT;To validate the kit,1994 clinical dog brain specimens submitted to our laboratory for rabies confirmation were detected in comparison with FAT and MIT. And the results by the three methods showed 100% agreement in fresh samples,while FAT/MIT was negative 1/2 samples in decomposed samples. These two studies showed that the kit was more sensitive than FAT and MIT and particularly useful for decomposed brain tissue.The test results of artificially infected mice brain tissues highlighted the utility of the developed kit in ante mortem diagnosis of rabies with higher sensitivity than FAT and MIT;Amongst the 2950 epidemiological monitoring saliva samples and 8 rabies saliva samples tested, both this kit and nested RT-PCR yielded 4/2950(0.14%) samples positive detection,showing 100% similarity,while this kit was positive in 3/8 samples, and nested RT-PCR positive in 2/8 samples According to the results above,these two studies proves that this kit can be used for early diagnosis of animals rabies and meet the demand of epidemiological investigation and diagnosis of animals rabies.Moreover, the results of test for the international inter-laboratory unknown samples showed that this kit has a complete consistency with DNA microarray for simultaneous detection and genotyping of lyssaviruses and nested RT-PCR and one step SYBR Green I real time RT-PCR for the detection of all lyssavirus genotypes.Thus it is demonstrated that the developed kit has good sensitivity and specificity and repeatability.The preclinical and clinical results for evaluation of several indexes and detection of clinical samples indicated that the method was very applicable in surveillance and diagnosis of animal rabies,which also provided reliable and supporting datas for its commercialization and widespread use. In addition, with the development of automation technology and its application, this kit has combined with the completely automatic sample grinder and nucleic acid Delivery protocol used for high-throughput screening.with the ability to detect thousands of samples every weekday.So it is not only suitable for diagnosis of single sample, but also suitable for large scale of epidemiological surveillance and rapid detection of animal rabies, which rendering this kit very applicable in elimination and control of rabies.