Establishment And Assessment Of Real Time RT-PCR Assays For Diarrhea-related Virus Detection

Background:Diarrhea diseases is a set of diseases which were caused by multiple factors and multiple pathogens, a common pediatric disease whose main features are more stool and characteristics changing, and a problem of worldwide public health. Pathogenic diarrhea include bacterial diarrhea, parasitic diarrhea and viral diarrhea. Viral diarrhea, among of them, is one of the main reasons for severe acute diarrhea. After Norwalk virus was found in1972, three RNA viruses that cause diarrhea have been successively discovered. Rotavirus、Calicivirus and Astrovirus have been identified as medically important pathogens. Although there are rare reports of massive outbreaks, Aichi virus can cause acute diarrhea. In addition, a few studies have shown that the Parechovirus and Enterovirus also can cause diarrhea. Viral diarrhea has been received concerns of WHO and national CDC in recent years, detection for multiple-virus should be carried out so as to provide scientific material for the diagnosis, prevention and control of diarrhea caused by diarrhea-related virus.Human rotavirus are members of the genus Rotavirus within the family Reoviruses, and the most common cause of severe viral diarrhea in infants and young children under the age of5worldwide. According to WHO2008estimates, about450000children aged <5years die each year from vaccine-preventable rotavirus infections; the vast majority of these children live in low-income countries. Rotavirus is composed of3layer structure protein RNA segments and is surrounded by the inner capsid proteins VP1, VP2and VP3. The VP6protein forms the middle layer of the virus capsid, while the outer layer is composed by VP4and VP7protein. Sequence characteristics of the gene segments encoding the VP7and VP4proteins are used to differentiate rotavirus G and P genotypes, respectively. While the genotypes G1、G2、 G3、G4、G9、[P4] and [P8] are the most prevalent worldwide, G1、G2、 G3、G9、[P4] and [P8] in this country. There are, so far, no effective drugs against rotavirus, vaccination is considered to be the most effective means to prevent rotavirus. Studies have shown that neutralizing antibodies against VP7/VP4have protective effect. So, the rotavirus classification should be carried out so as to provide scientific material for the diagnosis and the application of rotavirus vaccine.Objective:1. To establish and evaluate TaqMan real time RT-PCR method for detection and absolute quantification of diarrhea-related virus, to quickly determine the virus that causes diarrhea under the same reaction conditions, to provide technical reserves for dealing with public health emergencies.2. To establish and evaluate TaqMan multiplex real time RT-PCR method for detection and absolute quantification of GARV, to realize rapid detecting、quantitating and genotyping, to provide new method for rotavirus typing.Methods:With rotavirus-rechecked stool specimens in2011gene sequencing, the sequence of VP4and VP7be obtained; Nucleic sequences of diarrhea-related virus were obtained through the GenBank database. Specific target sequences of each genotype and diarrhea-related virus were determined by MAGE software analysis,. TaqMan probes and Primers were designed under the same reaction by Primes oftware. Blast program was used to preliminarily analyze the specificity of each primer and probe.Purpose fragment got T7promoter by connecting with pGEM-T Easy vector. RNA templates of target gene were synthesized by in vitro transcription. RNA standards were used to evaluate each real time RT-PCR assay, standard curves were established, sensitivity, specificity and reproducibility were evaluated. The practicability was preliminarily calculated by blind and clinical samples detecting.Results:1. Establishment of TaqMan real time RT-PCR assays for detection and absolute quantification of diarrhea-related virusRNA standards of each diarrhea-related virus were used to evaluate each singleplex real time RT-PCR assay, standard curves were established, the detection limits ranged from30to240copies/PCR. Cross-reactivity of primer-probe pairs for detection of each diarrhea-related virus and intestinal adenovirus were checked, the results showed that there were no significant amplification curves, which suggested high specificities of every primer-probe pairs. Repeated experients were employed to verify the stabilities of the method, and coefficient of variation were less than5%, which suggested well repeatability. Blind test results showed that the method can accurately determine the virus that causes diarrhea. Detection rate of RV、NV、 ASTV、EV、HPeV、SaV and Aichi is4.57%.17.39%.6.52%.7.61%、4.35%、9.78%and0.0%, respectively.2Establishment of TaqMan multiplex real time RT-PCR assays for detection and absolute quantification of RotavirusRNA standards of each genotype were used to evaluate each singleplex real time RT-PCR assay, standard curves were established, sensitivity were evaluated, the detection limits ranged from30to150copies/PCR. Cross-reactivity of primer-probe pairs for detection of each genotype, intestinal adenovirus, NV, SaV, ASTV and HPeV were checked, the results showed that there were no significant amplification curves, which suggested high specificities of every primer-probe pairs.RNA standards of each genotype were used to evaluate each multiplex real time RT-PCR assays, standard curves were established, sensitivity were evaluated, the detection limits ranged from30to240copies/PCR, and there were no significant differences compared with single-plex real time RT-PCR assay. under multiplex conditions, Repeated experients were employed to verify the stabilities of the method, and coefficient of variation were less than5%, which suggested well repeatability. Blind test results showed that the method can accurately determine the genotype of RV. Using rotavirus positive stool specimens, the method was preliminarily calculated. Comparing with the traditional method, genotype was able to accurately and fast quantitative determination by this method.Conclusions:TaqMan real time RT-PCR assays for detection and absolute quantification of diarrhea-related virus(Aichi、ASTV、EV、HPeV、RV and SaV) was established and evaluated. a rapid, sensitive and quantitative method. The method quickly determine the virus that causes diarrhea under the same reaction conditions, to provide technical reserves for dealing with public health emergencies.TaqMan multiplex real time RT-PCR assays for detection and absolute quantification of Rotavirus was established and evaluated, a rapid, sensitive and quantitative method. The method for Rotavirus detection realize rapid detecting quantitating and genotyping for major genotype in this country, to provide new method for rotavirus typing.