| Objective: 1. HEV ELISA kit effectiveness(including specificity and sensitivity) will be fully evaluated on our automatic diagnostic platform. 2. To ultimately establish a standardized diagnostic procedure and evaluation system from HEV RNA extraction to RT-PCR and one-step quantitative PCR. 3. To develope a RT-LAMP system to amplify HEV RNA to be used in front field.Materials and Methods: 1. Five distinct commercial HEV antigen or antibody ELISA Kit were used to parellally detect Serum samples, which were collected from Hepatitis E, Autuo immune disease, patients early infected by CMV or EB virus and healthy people, respectively. Sensitivity and specifity of each ELISA kit were evaluated and cmpared with each other 2. HEV cell culcture supernatant were used as neutralization antigen to verify whether the results is ture or not when inconsistency happened between different ELISA kits. 3. Fluorescence quantitative PCR for HEV RNA was designed covering genotype 1-4 HEV isolates, sensitivity and specifity were evaluated using clinical samples. HEV cultured cells system will be used to build serum plate through multiple proportions dilution methods with pathogen-free serum, HEV plasmid DNA reference standards will be created as quantitative PCR standards. 4. RT-LAMP was designed to amplify HEV RNA covering genotype 1-4HEV isolates, and performance of this assay including sensitivity and specifity were evaluated using clinical samples.Results: 1. Different HEV Ig M antibody ELISA kits have good consistenct results,while their diagnostic sensitivity is poor with not more than 85%, but their specifity is good enough with no cross recation were found in this study; Different HEV Ig G antibody ELISA kits have a good consistency with high diagnostic sensitivity(over90%) among patients. However, the detectiong results among other people excluding hepatitis e patients showed a great difference. For example, positive rate of people without Hepatitis E by Wantai kit is between 16.5% and 31.9%, that is 1.1% to 3.3%by Ke Hua kit. No false positive samples were found through neutralization inhibition test. 2. A real-time RT-PCR system was designed in this study, which can amplify genotype 1-4 HEV strains covering rabbit HEV suitable for serum and feces specimen. The minimum detection limit can be up to 25 copies/test, which is over 10 times high than that of RT-n PCR. The within-run and between-run imprecision at the103 and 106 copies/μl concentration levels is less than 2% and 3%, respectively. The study constructed the HEV plasmid standard and HEV RNA quality control serum plate, which is evaluated by stability assessment and can meet the preliminary requirements of clinical application. 3. A RT-LAMP assay was developed to detect HEV RNA in this study. Results showed that the detection limit of this assay reached as low as 5 copies/reaction and no cross-reactivity was observed with other hepatitis virus including hepatitis A, B and C virus. Furthermore, 233 clinical samples were retrospectively investigated with real-time RT-PCR, the nested RT-PCR, in parallel with this new assay. Results show that the RT-LAMP and Real time RT-PCR detection positive rate was significantly higher than that of RT-n PCR.Conclusion: 1. HEV Ig M and Ig G ELISA kits have their own merits, Kehua Ig G kit is suitable for clinical diagnosis and Wantai Ig G kit is better for epidemiological survey, combination of the HEV Ig M kits and Kehua Ig G kit is good to improve the efficancy of clinical diagnosis. Whether HEV antigen ELISA kits is necessary for clinical diagnosis is still uncertain, no support data were got in this study. 2.Fluorescence quantitative PCR for HEV RNA was developed in this study, and nucleic acid standards and quality control serum plates were built and evaluated to solve the core problem of lack of standardization of laboratory diagnostic procedures and evaluation system for clinical application. 3. using a combination of sensitivity,specificity and evaluation of clinical samples, we were able to reported that a new diagnositic way(RT-LAMP) is provided to detect HEV RNA, which has great chance to be used in front field, considering its great merits. However, the reliability of this assay should be further evaluated by a large-scale investigation due to the high genetic viaribilty especially from different geographic regions... |
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