Development And Evaluation Of A Loop-mediated Isothermal Amplification Method For Rapid Detection Of HCMV DNA In The Serum Of Pregnant Women

Human cytomegalovirus (HCMV) was the most frequent and serious cause of active infections of pregnant women and intrauterine infections, could be transmitted during pregnancy from the mother with active infection to the fetus by vertical transmission or uplink infection and cause serious damages, such as abortions, stillbirths, deformities and multiple organs dysfunction after birth. Conventional ELISA could not meet the clinical requirements for its low sensitivity and specificity, FQ-PCR was expensive and required special equipments. The aim of our study was to establish a highly sensitive, rapid, specific and low-cost method to test HCMV DNA in the serum of pregnant women and to explore its clinical valueLoop-mediated isothermal amplification (LAMP) was applied to quickly detect HCMV DNA in the serum of pregnant women. Two specific pairs of LAMP primes were designed that are for the recognition of a conservative IE1 gene in HCMV genome. The reaction was carried out at 60℃for 1 hour and the products were separated by 2.5% agarose gel electrophoresis.The results optimizing the factors which could affect experiment showed temperature was the most important factor and the concentration of magnesium ions and betaine had little effect on the LAMP reaction. In order to evaluate the specificity of the HCMV primers, HCMV, herpes simplex virus type-1(HSV-1), HSV-2, and human genome DNA were amplified by LAMP method. Only amplified HCMV demonstrated the typical ladder patterns; no LAMP product was detected in reactions performed with other three viral DNAs. To further confirm products specificity, The HCMV amplified products were digested with Bsu36Ⅰrestriction endonucleases and its sizes analyzed by gel electrophoresis. The sizes of the fragments generated were approximately 116bp and 104bp and in good agreement with the predicted sizes. The sensitivity of LAMP method was also determined. Serial dilutions of the serum DNA were used to determine the detection limit of HCMV LAMP, which was 102copies/ml as determined by 2.5% agarose gel electrophoresis.After these initial validation studies, 126 samples of serum from pregnant women were tested for HCMV by LAMP method. The results of LAMP were compared with Fluorescent quantitative PCR (FQ-PCR) and enzyme-linked immunoassay (ELISA).The positive cases was detected in 42 of 126 samples by LAMP, and in 44 by FQ-PCR, and ELISA for 14. The ELISA and LAMP technique showed 31.8% and 95.4% positive concordance with FQ-PCR, respectively. There was no significant difference (P>0.01) was observed between LAMP and FQ-PCR, but difference is significant (P<0.01) between ELISA and FQ-PCR.In conclusion, our results indicated that LAMP method applied to detection of HCMV DNA in the serum of pregnant women had good sensitivity and specificity that were similar with FQ-PCR. Its accuracy was far higher than conventional ELISA technique for the detection of HCMV infections. After further optimization, improvement and extension, the technology was expected to play an important role to prevent birth defects and infectious diseases prevention.