Development And Application Of Loop-Mediated Isothermal Amplification Methods For Detection Of Enteroviruses

Enteroviruses (family Picornaviridae) are among the most common viruses infecting humans worldwide, in which human enteroviruses (HEVs) including coxsackieviruses, echoviruses, polioviruses and newly discovered enteroviruses. The spread of infections is mainly through the faecal-oral and oral-oral route. For example, infection is transmitted through the faecal-oral route by contact with water, food and soil contaminated with infected feces. Enteroviruses infect millions of people worldwide each year, resulting in a wide range of clinical outcomes. The traditional assay for detection of enteroviruses is virus isolation in cell culture, followed by analysis with neutralizing antisera. The cultures yield a positive result within the first week, and the assay is time-consuming and expensive. Therefore, efficient, sensitive, rapid, and simple methods for the detection of enteroviruses are needed.Notomi T etc invented loop-mediated isothermal amplification (LAMP) in 2000, which owned higher sensitivity and specificity, further shorten the amplification time, improved the amplification efficiency, and reduced the equipment and test condition requirements.Thus, we developed new methods which were highly specific and sensitive to detect enteroviruses. Up to now, we had successfully applied the LAMP assay for detecting enteroviruses in stool samples from hand, foot and mouth disease (HFMD) patients, drinking water source samples and food packaging samples, and the LAMP assay for detecting poliovirus in drinking water source samples also showed a good result.Objective:Developt two loop-mediated isothermal amplification (LAMP) assays, one is to detect enteroviruses, and the other is to detect poliovirus only. Apply the two assays to detect enteroviruses and poliovirus in samples. Apply real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect poliovirus in the drinking water source samples of Tianjin.Methods:1 Collected and analyzed genetic sequences of enteroviruses from the GenBank, the target-specific sequence was obtained, and a set of 4-specific primers was desigened. The amplication took place relying on Bst DNA polymerase under isothermal conditions. Optimized the reaction system by adjusting the concentration of Mg2+, betaine, dNTPs and the reaction temperature. Evaluated the specificity and sensitivity of the assay. Applied the optimal LAMP assay to detect enteroviruses in stool of HFMD patients, drinking water source and food packaging samples. The products could be detected by the turbidity with the naked eyes, or by a color change after addition of SYBR Green I dye to the products. The products also could be analyzed by restriction digestion with a restriction enzyme, NlaIV, or through carrying out sequence analysis.2 Collected and analyzed genetic sequences of poliovirus from the GenBank. A set of specific primers for poliovirus was desigened. Optimized the reaction system, evaluated the specificity and sensitivity of the assay. Applied the optimal LAMP assay to detect poliovirus in 9 drinking water source samples of Tianjin city. The products could be observed through white precipitate by the naked eyes or color changes by addition of SYBR Green I. Analyzed the LAMP-amplified products by restriction digestion with a restriction enzyme, HpyCH4III, and then 2 LAMP products randomly selected were sent to carry out sequence analysis.3 At the same time, applied a real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect poliovirus in the 18 drinking water source samples of Tianjin.Results:1 The LAMP method for detection of enteroviruses was carried out within 1 to 1.5h. The LAMP assay had high specificity for detection enteroviruses, including EV71, CoxA16, CoxB3, CoxB5 and Echo30. The LAMP detection limit was 101 copies/μL, which was as sensitive as PCR for enteroviruses detection. Detection enteroviruses in samples indicated that all of the water samples and 18 of 19 fecal samples showed positive results, the positive rates were 9/9 (100%), 18/19 (94.7%), respectively, while all of the food packaging samples showed negative results. After restriction digestion with NlaIV, two sizes of fragments, 87 bp and 103 bp, mainly generated,which was consistent with the expected results. All of the 10 cases products sent to carry out sequence analysis aligned well with the 5′UTR of enteroviral genome in the GenBank, among which 2 products were coxsackievirus A16 mostly like, and 8 products were enterovirus 71 mostly like.2 The method developed for detection of poliovirus was carried out within 1 h to 1.5h. The LAMP assay had high specificity for detection poliovirus only. The detection limit was 101 copies/μL, which was equal to the PCR method, while the LAMP assay was more rapid, convenient and simple equipment required. Detection poliovirus in samples indicated that 8 of 9 water samples showed positive results. After restriction digestion with HpyCH4III, the generated fragments converged mainly in the size of 97 bp, which was consistent with the expected results. All of the 2 cases products sent to carry out sequence analysis aligned well with poliovirus genome in the GenBank.3 The viral recovery rate using Ultracel-100k centrifugal filter devices was 34.83%. RT-LAMP detecting poliovirus applied to the 18 water samples indicated that the concentration of poliovirus in the positive samples ranged from 7.5×105 to 1.1×108 copies/L.Conclusion:In this study, we established the LAMP assay for detection of enteroviruses and the LAMP assay for detection of poliovirus successfully, which were simple, rapid response, high specificity and sensitivity. So the two assays provide the technology platform for the enteroviruses and poliovirus detection in samples, having a good application prospect in fast detection.