Development And Application Of RT-LAMP Technique In Detection Of Enterovirus 71 Gene

PrefaceEnterovirus 71 (EV71) is a member of the genus Enterovirus in the family Picornaviridae.It is a single-stranded positive-strand RNA virus and it's genome consisted by more than 7400 nucleotides. In 1974 EV71 was isolated the first time from the stool samples of baby with the central nervous system diseases in Californi froma. Now EV71 has caused a dozen worldwide outbreak and spread to the world, particularly in the Asia Pacific region in recent years, the incidence has increased. EV71 was first discovered in Shanghai In 1981, so far in Beijing, Hebei, Tianjin, Fujian, Jilin, Shandong, Hubei, Xining, Guangdong, Xiamen, Liaoning Province, Wuhan, Hong Kong and other 20 provinces have reported.EV71 has a certain tissue tropism in regard to the spinal cord anterior horn cells, as the most common Non-polio Fmterovirus(NPEV) that causes Acute Flaccid Paralysis(AFP). In recent years EV71 is more related to the outbreaks of Hand, foot and mouth disease(HFMD), and causes heavie clinical symptoms.Some patients may be associated with aseptic cerebro-spinal meningitis, encephalitis, myocarditis, and brain sequelae of symptoms such as paralysis or even death. Therefore, the rapid and specific detection methods are particularly important, especially diagnose severe disease caused by the EV71 infection.Loop-mediated isothermal amplification (LAMP) is established as a new nucleic acid amplification by Notomi. in 2000. Its principle is to use a kind of chain replacement DNA polymerase (BstDNA polymerase) and two pairs of specific primers to identify specific target sequences on six separate regions, in isothermal conditions (65℃or so) holding one hour, you can comple the amplification of nucleic acid. Reaction results can be amplified by-product of the precipitation of magnesium pyrophosphate to determine turbidity, also can be added SYBR Greenâ… , ethidium bromide (EB) or other nucleic acid dyes for coloring to watch. The advantage is specificity, amplification efficiency, responsive and easy to operate.The technology has been applied to a variety of bacterial and viral gene detection by domestic and foreign scholars because of its specificity, amplification efficiency, responsive, easy to operate. The report about the technology used in detection of EV71 gene has not yet retrieved. In this paper, three pairs of specific primers were designed to capsid protein VP1 gene sequence of EV71. RT-LAMP amplification system was optimized for the detection of the target sequences, and its specificity and sensitivity were verified through experiments. The examinations have carried out to detect EV71 gene in the specimens of clinical diagnosis HFMD patient through this technolog in Liaoning Province. The result of RT-LAMP was compared with the result of reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescence quantitative PCR (Real-time PCR) methods. The purpose is to seek fast, simple and practical new methods that applied for clinical diagnosis of EV71 infection.Methods1. Primer design and synthesizeThe VP1 genetic sequence of EV71was downloaded from the GenBank and analysed by DNAStar analysis software. Six primers were selected by LAMP Primer Explorer 4 primer design software of Japanese eiken company in the sequence of the conserved region and analysed by BLAST,identified as specific primers of EV71. The primers were synthesized by the Shanghai Health Industry company and dissolved by ddH₂O. The primers were saved for use in-20℃refrigerator.2. Specimen collection and processingWe collected 297 cases HFMD patients through clinical diagnosis in Liaoning Province and gathered stool or pharynx specimens of those patients. The 239 specimens were inoculated by Vero cells. If the culture produced cytopathic effect (CPE), we collect the virus fluid and extract nucleic acid.The other 58 specimens were directly extracted nucleic acid without pre-treatment. 3. Nucleic acid extraction and preservationThe nucleic acid extraction was carried out by using QIAGEN's RNeasy mini kit nucleic acid extraction kit. we extracted RNA of specimens according to procedures provided by the company and saved immediately RNA to backup in-70℃condition.4. RT-LAMP real-time monitoringIn order to establish optimal reaction conditions, we have been optimized RT-LAMP reaction system. In the reaction system by adding SYBR Green I fluorescent dye we can monitored the process of RT-LAMP amplification. The condition of RT-LAMP amplification is 60℃for 80 minutes in the fluorescent quantitative PCR (Real-time) instrument.We detected amplified product through the dissolved curve analysis. Finally, we analyzed test data.5. Sensitivity and specificity of RT-LAMPTo evaluate the specificity of the technology, we carried out RT-LAMP amplification for the same time using designed specific primers. The amplification templates were rotavirus, Coxsackie virus A16, norovirus, Sapporo viruses and EV71. The RNA template of EV71 confirmed by RT-PCR method was diluted into a 10-fold serial dilutions of 1.0×10-1～1.0×10－6 for sensitivity testing. At the same time sensitivity of RT-LAMP was compared with the Real-time PCR and RT-PCR method.6. Specimens testingWe detected the above samples through RT-LAMP assay, and compared test results at the same time with the result of RT-PCR and Real-time PCR methodResults1. Specificity of RT-LAMP methodThe there pairs of specific primers designed in this paper were response with the EV71's RNA only. It has not a cross-reaction with rotavirus, Coxsackie virus A16, norovirus, Sapporo, such as viral nucleic acid. The primer indicated has a relatively high specificity. And the analysis to dissolution curve of PCR products verified the specificity of amplification.2. Sensitivity EV71 RNA of clinical samples was diluted to 1.0×10-1～1.0×10-6 dilutions.The result of RT-LAMP showed a typical LAMP amplified banding pattern, the signal was weakened gradually, and the lowest detection density was 10-5. But thelowest detection density of RT-PCR and Real-time method were 10-4. It showed that RT-LAMP will bring us the highest sensitivity between the three ways.3. The test results of SpecimensFor cell cultures of the throat specimens there wasn't any significant difference between the abilities of the three methods in detect of EV71 gene (p>0.05). For cell cultures of the urine specimens there wasn't any significant difference between RT-LAMP and RT-PCR methods (p>0.05),but it was higher than Real-time PCR method (p<0.05).For untreatment clinical samples,the analyse result of total specimens indicate that between RT-LAMP and Real-time PCR methods (p>0.05),but it was higher than RT-PCR method (p<0.05). The analyse result of throat specimens indicate that there wasn't any significant difference between the abilities of the three methods in detect of EV71 gene (p>0.05). But the analyse result of urine specimens indicate that between RT-LAMP and Real-time PCR methods (p>0.05),but it was higher than RT-PCR method (p<0.05).DiscussionFirst, we chosed VP1 protein when primer designing. VP1 protein has the largest number of type-specific neutralization sites, which will provide the most reliable information about molecular epidemiology and is the current best regional in genotype detect. In the region three pairs of primers were designed and it can specifically identified the eight specific areas of target sequences, and only when the primer pairs are mated strictly with the eight regional a large number of target sequences will be amplified. So the reaction has high specificity nature and it is not be effected by the existence of non-target sequences of DNA. Second, in the specificity testing of RT-LAMP EV71 gene was detected successfully, but rotavirus and other erovirus was not amplified. The specificity of RT-LAMP reaction was further proved by the dissolution curve analysis of PCR products.EV71 RNA of clinical samples was diluted to 1.0×10-1～1.0×10-6 dilutions.The result of RT-LAMP showed a typical LAMP amplified banding pattern, the signal was weakened gradually, and the lowest detection limit was 10"5. But the lowest detection limit of RT-PCR and Real-time methods were 10-4. It showed that the sensitivity of RT-LAMP method was the highest between the three methods.Through theχ2 tests for cell cultures, it was founded that the throat specimens there wasn't any significant difference between the abilities of the three methods in detect of EV71 gene (p>0.05). For cell cultures of the urine specimens there wasn't any significant difference between RT-LAMP and RT-PCR methods (p>0.05),but it was higher than Real-time PCR method (p<0.05). The probable cause was that the virus content of throat specimens are highen than urine specimens.For the detection of a large quantity virus the fluorescence signals were not detected by Real-time PCR method.For untreatment clinical samples,the analyse result of total specimens indicate that between RT-LAMP and Real-time PCR methods (p>0.05),but it was higher than RT-PCR method (p<0.05). The analyse result of throat indicated that there wasn't any significant difference between the abilities of the two methods in detect of EV71 gene (p>0.05). The analyse result of urine specimens indicated that it was higher than RT-PCR method (p<0.05). The probable cause was that the numble of throat specimens was few,when statistics analyses were carried out the error occurred.ConclusionThe RT-LAMP technique established in this study get a better result for the detection of stool and throat specimens of EV71 gene. It has a good consistency with the Real-time PCR method, what's more, its positive detection rate is higher than conventional RT-PCR method. The detection process reflected its rapid, sensitive, specific and low cost.