Establishment Of Three RPA Detection Methods And Preparation Of Positive Reference Substance

Tick-borne encephalitis virus(TBEV), is a member of the genus Flavivirus, and is transitted by ticks. Meanwhile, TBEV has three subtypes: Western European subtype, Siberian subtype, Far Eastern subtype. Tick-borne encephalitis virus is a hazard to Tick-borne encephalitis which is a viral infectious disease involving the central nerous system. Long-lasting or permanent neuropsychiatric sequelae are observed in 10 to 20 percent of infected patients, that indicates a bad prognosis. Recently, the number of reported cases has been increasing in northern forests of our country.Ebola virus(EBOV), causes a severe and ofen fatal hemorrhagic fever in humans and other mamals, known as Ebola hemorrhagic fever. Ebola virus is the single member of the genus Ebolavirus, family Filovivirus. Meanwhile, EBOV has five subtypes: Zaire subtype, Sudan subtype, Ivory Coast subtype, Reston subtype, Bundibugyo subtype. Zaire Ebola virus and Sudan Ebola virus are highly pathogenic and lethal to human and non-human primates. Zaire Ebola virus is the cause of the 2013-2015 Ebola virus epidemic in West Africa, which has resulted in at least 28,638 suspected cases and 11,315 confirmed deaths. Because of its high mortality rate, and there is no specific vaccine or treatment for the disease, EBOV is also listed as World Health Organization Risk Group 4 Pathogen.Middle East respiratory syndrome(MERS), also known as camel flu, is a viral respiratory infection caused by the Middle East respiratory syndrome coronavirus(MERS-CoV). Symptoms range from mild to severe. They include fever, diarrhea, cough and shortness of breath. Disease is typically more severe in those with other health problems. There is no specific vaccine or treatment for the disease. Just over 1000 cases of the disease have been reported as of May 2015. About 40% of those who become infected die from the disease. MERS-CoV is a new member of the beta group of coronavirus(Betacoronavirus). It was first reported in 2012. As of July 2015, MERS cases have been reported in over 21 countries, including the United States, the United Kingdom, South Korea and Mainland China, arousing people’s concern and panic.Therefore, early diagnosis and prompt treatment of viral infectious diseases play an important role in the rehabilitation and prognosis of the patients. The detection of the virus is a prerequisite for the exact diagnosis of the disease. At present, the laboratory and clinicaldiagnosis of viral infection mainly depends on virus cell culture, specific antigen or antibody detection, red cell agglutination reaction and viral nucleic acid detection. In recent years, with the development of the polymerase chain reaction technology, the importance of the rapid detection technology of nucleic acid is increasing.A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction(PCR). It monitors the amplification of a targeted DNA molecule during the PCR, and not at its end, as in conventional PCR. The real-time polymerase chain reaction can also be used quantitatively. Two common methods for the detectin of PCR products in real-time PCR are: nonspecific fluorescent dyes that intercalate with any double-stranded DNA; and sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence. In the virus detection, designing specific primers and probes can realize our rapid detection of viral nucleic acid.However,viral infectious diseases usually outbreak in remote areas, in which instruments and reagents are lacked. It also reflects the limitations of RT-qPCR for field. Therefore, we try to find a rapid detection technique for field and remote areas in rapid viral detection field.Recombinase Polymerase Amplification(RPA) is a novel isothermal nucleic acid amplification technology. The isothermal amplification of specific DNA is achieved at 37-42! within 10-15 min. In RPA, the recombinase complexes perform the binding of opposing oligonucleotide primers to template DNA and the DNA polymerase perform the extension. The key to RPA is the establishment of a dynamic reaction environment that balances the formation and disassembly of recombinase-primer filaments, for that we employ T4 uvs X, bindings cooperatively to oligonucleotides in the presence of ATP. By adding a reverse transcriptase enzyme to the reaction, it can detect RNA. The RPA products observation can be realized by using the acrylamide gel electrophoresis. Also, by adding a probe to the reaction, it can become a real-time RPA. Because it is isothermal, RPA reaction needs much simpler equipment than PCR, which needs a thermal cycler. This makes RPA an excellent candidate for developing low-cost, rapid, point-of-care molecular tests.The RPA technology was established by Olaf Piepenburg、ColinH.Williams、Derek L.stemple in the University of Cambridge, and has existed only 10 years, that can be defined as a novel isothermal amplification for DNA and RNA. The technology overcomes the technical difficulties, which are the absence of temperature-regulating equipment in theremote field. The viral nucleic-acid detection can be achieved by using a 1kg fluorescence detector with a laptop or an incubator with the strips.Armored RNA virus-like particle(AR) technology is technique of preparation for RNA qulity control. This kind of RNA qulity control is more secure than inactivated virus, and more accurate than artificial plasmid. The principle is that E.coli bacteriophage MS2 coat protein gene and our target gene were cloned into the expression vector by genetic engineering methods. Meanwhile, our target sequence can be transcripted into RNA, which can bepackaged into MS2 coat protein, finally, that is called armored RNA virus-like particle. Apparently, these paticle can resist the digestion of RNase.We developed rapid RT-qRPA assays for detection of Tick-borne encephalitis virus, Zaire Ebola virus and Middle East respiratory syndrome coronavirus, and evaluated the specificity and sensitivity of all of the assays by comparing with RT-qPCR assays. Firstly, for TBEV assays, we designed primer and probe targeting the 3’non-coding region of the TBEV genome. Meanwhile, we developed RT-RPA assay, 3 kinds of two-step RT-qRPA assays with 3 kinds of reverse transcriptase, 5 kinds of one-step RT-qRPA assays with 5 kinds of reverse transcriptase. We evaluated the sensitivity of the assays with TBEV virus and TBEV-AR virus-like particles. We verified the specificity of the assays with flavivirus RNA other than TBEV. Finally, we detected 54 clinical suspected samples,using one-step RT-qRPA assay reported in this thesis and compared the results with the RT-qPCR assay. Secondly, for ZEBOV assays, we designed primer and probe targeting the NP region of the ZEBOV genome. Meanwhile, we developed two-step RT-qRPA assay. We evaluated the sensitivity of the assays with the artificial plasmid. We verified the specificity of the assays with hemorrhagic fever syndrome virus RNA. Thirdly, for MERS-CoV assays, we designed primers and probes targeting the upE region and the N protein region of the MERS-CoV genome. Meanwhile, we developed 2 kinds of one-step RT-qRPA assays with 2 kinds of reverse transcriptase. We evaluated the sensitivity of the assays with MERS-CoV-upE-AR virus-like particles and MERS-CoV-N-AR virus-like particles. We verified the specificity of the assays with virus RNA other than MERS-CoV.The two-step TBEV RT-qRPA assay could detect 10-2TCID50 for the virus.; the one-step RT-qRPA assay could detect 1.8copies of the TBEV-AR virus-like particles. The results of the detection of the 54 clinical suspected samples with the one-step RT-qRPA assay had no statistical difference with the results with the RT-qPCR one. The ZEBOV two-step RT-qRPA assay could detect 100 copies of the artificial plasmids. The MERS-CoV-upERT-qRPA assay could detect 4 copies of the MERS-CoV-upE-AR virus-like particles; the MERS-CoV-N RT-qRPA assay could detect 1 copie of the MERS-CoV-N-AR virus-like particle. In addition, all of the assays have high specificity. Under the electron microscope, the 25 nm spherical virus-like particle were visible.Ultimately, because of the distribution of the tick borne encephalitis reported cases was always in the northen remote forest areas of our country, we develop the RPA assay for the detection of the TBEV, whose sensitivity was higher than the RT-qPCR assay. In addition, for the two emerging infectious diseases ZEBOV and MERS-CoV, which attracted the scare of people, the RPA assays were developed, which performed as well as the RT-qPCR assay, or better than the RT-qPCR one.