Establishment of a two-step duplex fluorescence quantitative PCR method for simultaneous detection of Giardia lamblia and Cryptosporidium parvum
LI Jia1, 2, 3, HUANG Da-na2, ZHANG Xiao-min2, NIU Cong1, 2, 3, WAN Cheng-song1, 3, ZHANG Ren-li1, 2
1.School of Public Health, Southern Medical University, Guangzhou 510515,China; 2. Shenzhen Center for Disease Control and Prevention, Shenzhen 518055,China; 3. Guangdong Province Key Laboratory of Tropical Disease Research, Guangzhou 510515,China
Abstract：Aimed to establish a two-step duplex fluorescence quantitative PCR method for simultaneous detection of Giardia lamblia and Cryptosporidium parvum. Specific primers and TaqMan probes were designed according to the gdh gene sequence of Giardia lamblia and the cowp gene sequence of Cryptosporidium parvum. Then we constructed the standard plasmids. The reaction system and conditions were determined after optimizing the concentration of the primers and probes. The sensitivity, specificity, stability and repeatability of the method were evaluated. What’s more, we compared this established method with gold immunochromat ographic assay by detecting iatrogenic diarrhea samples suspected of "two insects" infection to evaluate its practical application value. The established method could specifically detect Giardia lamblia and Cryptosporidium parvum, and had no amplification curves for other non-target species, which showed good specificity of the method. The sensitivity of the method for simultaneous quantitative amplification of positive plasmids pMDTM19-T-GIA and pMDTM19-T-CRY was 45.1 copies/μl and 52.8 copies/μl, respectively; the standard curve showed good linearity (R2=0.99) between 109 copies/μL101 copies/μL. The coefficient of variation for both intra- and inter-assay replicates was less than 5%. At the same time, the coincidence rate between the established method and gold immunochromat ographic assay is 100%. Resultsof this study suggested that the two-step duplex fluorescence quantitative PCR method not only can be used to detect Giardia lamblia and Cryptosporidium parvum simultaneously, quickly, sensitively and specifically, but also has good practical application value.
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2020, Vol. 36 
