Establishment And Application Of A Loop-mediated Isothermal Amplification Method For Detection Of Herpes Simplex Virus DNA

Objective:To set up a new method detecting HSV DNA in serum of pregnant women,with high sensitivity,specificity and low-cost,and the clinical value of this technique were evaluated as well.Methods:We designed two specific LAMP(Loop-mediated isothermal amplification) primers for HSV-â… and HSV-â…¡respectively,and established the reaction system to detect HSV by LAMP.To determine the best conditions of this method,we optimized factors influencing LAMP,and assessed sensitivity and specificity of this method.On the basis,96 samples of peripheral blood from pregnant women were detected.At the same time,these specimens were also detected by ELISA and FQ-PCR, and the results were compared.Results:The results of optimized experiment indicated that temperature was the strongest impacting factor and the concentration of betalne was also very important in our established system.To contrast,the concentration of magnesium ions and other factors were less.The specificity experiment showed that primer for HSV-â… - specific only amplified HSV-â… DNA,and HSV-â…¡-specific primer only amplified HSV-â…¡DNA, no other DNAs could be amplified.Enzyme digestion further proved the specificity of amplification.The sensitivities of HSV-â… and HSV-â…¡specific LAMP methods,both reached 10 copies per reation.After these initial validation,96 serum samples from pregnant women were detecteded by our established LAMP method.Sixteen positive cases for HSV-â…¡were detected,while nine HSV-â… positive cases were detected. Compared with FQ-PCR,which was a golden standard in clinical test,the LAMP showed more 94%concordance,there was no significant difference(P>0.01),but difference is significant(P<0.01) between ELISA and FQ-PCR. Conclusion:The results indicated that the LAMP methods which we established for detecting HSV DNA had high sensitivity and specificity,which were similar with FQ-PCR and higher than conventional ELISA.Further more,this method is charactersied by simple operation,rapid procession,easy to judge and low-cost.After further optimization the technology was expected to play an important role on diagnosing infection of HSV and preventing birth defects and infectious diseases.