SYBR Green Real Time PCR Assay For Rapid Detection Of Plasmodium Falciparum And Plasmodium Vivax

Objective:To develop a SYBR Green real-time PCR assay to rapidly detect Plasmodium falciparum and Plasmodium vivax. And to use the established methods for preliminary clinical application.Method:1. Two pairs (Three strips) of outer primers and Two pairs (Three strips)of inner primers were designed according to the sequences of the18S rRNA of the two kinds of Plasmodium. A multiplex-nested PCR for detecting Plasmodium falciparum and Plasmodium vivax was established. Specificity and sensitivity of the assay were assessed. Patients'blood samples were detected with the multiplex-nested PCR.2. The two kinds of Plasmodium were detected by SYBR Green based real time PCR with the analysis of dissociation curve (DC) and melting temperature(Tm), respectively. Specificity, sensitivity and repeatability of the assay were assessed. Patients'blood samples were detected with the SYBR Green based real time PCR.Result:1. The sizes of amplification products of multiplex-nested PCR amplifying genomic DNA of Plasmodium falciparum and Plasmodium vivax were162bp and112bp respectively. The clinical sensitivity was97.30%and the specificity was5.88%, and the limit of detection was101opies/?1.2. The standard curves of SYBR Green real time PCR showed fine linear relationship between Cycle threshold and template concentration, the amplification efficiency was101.884%and limit of detection was101copies/?l. It took only3hours from DNA extraction to completion of PCR. And it showed good reproducibility. The results showed that the assay had no cross-reaction with Plasmodium malariae and Plasmodium ovale.54patients' blood samples were detected successfully.Conclusion:1. The multiplex-nested PCR combines both sensitivity and specificity for detecting Plasmodium falciparum and Plasmodium vivax simultaneously. It can be used for detection and identification of Plasmodium falciparum and Plasmodium vivax in Primary Sector.2. The SYBR Green based real time polymerase chain reaction is suitable for detection and identification of Plasmodium falciparum and Plasmodium vivax with high sensitivity, low-cost, low risk of contamination, high-throughput, and without fluorescence probe. There are11figures,7tables and53references.