Rapid Pathogen Detection Technology And Establishment Of Biobank

Owing to the global climate warming, accelerating of urbanization, rapid development of tourism and international trading, and the changing of the ecological environment, the incidence of acute infectious diseases are rising in recent years, such as the outbreak of H5N1 in 1997 in Hong Kong, the SARS epidemic in 2003, the HFMD epidemic in 2008 in Fuyang city Anhui province as well as the outbreak of H1N1-pdm09 virus in 2009, the prevalence of infectious diseases increase frequently. So it’s crucial to develop new detection technologies for emerging pathogens surveillance and monitoring needs. In this thesis, we used real-time RT-PCR and LAMP technology to detect H1N1-pdm09 and EV71 virus, supplemented with other techniques are employed to investigate the following topics.1. Rapid nucleic acid detection method of H1N1-pdm09 virus and molecular characterization of the HA gene segmentH1N1-pdm09 virus was first detected in an outbreak of "swine flu" epidemic in Mexico in March 2009 which spreaded quickly across worldwide. The first confirmed case was reported in May 11,2009 in Mainland China, since then, a total of 128,000 infected cases and 805 death cases were reported by Chinese government. In this study, real-time RT-PCR and RT-LAMP assays were developed for the detection of H1N1-pdm09 virus and the molecular characterization of HA gene segment was also analyzed. We report the design and characterization of a novel set of primers to be used in a real-time RT-PCR assay for detecting the H1N1-pdm09 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the H1N1-pdm09 virus and are different from those targeted by the WHO-recommended primers. The real time RT-PCR assay with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assay with our primers were 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples,32 were detected to be positive using the real-time RT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers, these results suggest that the real-time RT-PCR assay with the newly designed primers represent a highly sensitive assay for diagnosis of the H1N1-pdm09 virus infection. At the same time, we developed RT-LAMP assay for the detection of H1N1-pdm09 virus in clinical samples, our results showed that specificity and sensitivity of the RT-LAMP assay was same as real-time RT-PCR assay, at the same time, we successfully developed visualization LAMP detection method which can be determined by the naked eyes, so it’s very useful for clinical diagnosis and prevention and controlling of emerging pathogens in poor and remote areas. The HA gene nucleotide sequence of H1N1-pdm09 virus (A/Taizhou/09/2009) which was isolated in Taizhou city was firstly determined and the molecular characterization of HA gene segment was analyzed. The HA gene segment was amplified by RT-PCR and cloned into pMD-18T, it’s sequenced by Invitrogen Shanghai. The nucleotide and deduced amino acid sequences were compared with the homologous regions of other influenza viruses isolates. Results showed that the predominant influenza virus strain circulating in Taizhou city was homology with other regions during 2009-2010 influenza seasons.2. Detection of EV71 by real-time RT-PCR assay and molecular characterization of the VP1 gene segmentHand, foot and mouth disease (HFMD) is a common childhood infectious diseases caused by a variety of human enteroviruses which occurred mostly in children under 5 years old, it’s belonged to class C infectious disease in our national statutory report system. Coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are the most common pathogens causing HFMD and EV71 becomes more popular in the Asia-Pacific region in recent years. In 2008, HFMD epidemic was outbreak in Fuyang city, Anhui province, a total of 1165 cases of severe cases which accounting for 0.24% of the total number (489,073) of reported cases, including 126 death cases. In this study, we established the detection assay of EV71 based on real-time RT-PCR method and molecular characterizations of VP1 gene segment were also studied. We designed and selected a set of probes and primers which located in the conserved regions of VP1 gene of EV71 viruses. Research results showed that the newly developed assays were more sensitive than conventional PCR recommended by the Ministry of health of China. At the same time, we detected 110 clinical suspected HFMD samples using our newly developed assays comparing with conventional PCR,48 were detected to be positive using the real-time RT-PCR assays with our designed primers, while only 31 were positive by the assays recommended by the MOH. These results further suggest that the real-time RT-PCR assays with the newly designed primers represent a highly sensitive assay for diagnosis of the EV71 virus infection. We isolated 9 strains of EV71 viruses from clinical samples collected from Nanchang city in Jiangxi province and Ezhou city in Hubei province. The VP1 gene segment of isolated EV71 viruses were amplified by RT-PCR and cloned into pMD-18T, it’s sequenced by Invitrogen Shanghai. The nucleotide and deduced amino acid sequences were compared with the other EV71 strains. Analysis results indicated that the predominant EV71 strain circulated in mainland China was C4 subtype at recently, there are also a few subtype A EV71 strains cocirculated in some regions of China, our results were consistent with reported by other Chinese scholars.3. The establishment of the viral hepatitis biobank and antiviral-resistant HBV mutations detection platformLiver diseases are common and frequently-occurring in Mainland China where is a high incidence area of hepatitis B virus, World Health Organization released data shows that there are 140 million hepatitis B and C virus carriers in China which accounting for 28% of the global; including 32 million chronic active liver disease patients,400,000 people died of viral liver disease each year; in addition, these exist 297,000 liver cancer patients which accounting for more than half of the total global patients with hepatocellular carcinoma (55%). "Chronic hepatitis B prevention and treatment guidelines" (2010) clearly pointed out that the treatment of chronic hepatitis B patients including antiviral, immune regulation, anti-inflammatory, anti-fibrosis and symptomatic treatment, and among all of these, antiviral treatment is the key point, as long as the indications and conditions permitting, it should be carried on the standard antiviral treatment. When HBV patients were resistance to certain anti-viral drugs, HBV genome had some clear and definite mutations, and genotypic resistance appeared tend to be 1-3 months before phenotypic resistance, so it’s necessary to detect HBV genotypic resistance in clinical to help doctors adjust the treatment plan timely to delay or prevent the occurrence of phenotypic resistance. At present, Theories thought that the emergence of nucleoside analogues resistance were due to mutations of the polymerase gene of HBV and the polymerase gene variant were multiplies. So we combined with Liver Department of People’s Hospital of Taizhou City established HBV anti-viral resistant mutation detection platform to detect HBV resistant mutations and give scientific guidance for clinical medication. At the same time, we also successfully developed a one-step amplification of HBV full-length genome sequences which can be used for HBV phylogenetic and genetic evolution analysis. At last, we successfully established biobank of the hepatitis virus samples of the Taizhou area, the biobank has collected nearly 100 samples with clinical data traceable, and the biobank will continue to be expanded which are very fundamental for follow-up studies.