Establishment of a SYBR Green real-time fluorescent PCR assay for the detection of filarial
Objectives To establish a SYBR Green real-time fluorescent polymerase chain reaction(PCR)which can detect filarial rapidly and sensitively.To provide laboratory basis for rapid screening and clinical diagnosis of filariasis.Methods Specific primers were designed to target the conserved region of filarial genome and then the real-time fluorescent PCR was established by optimizing both the concentration of SYBR Green,primers and the amplification conditions.The lower limit of detection,specificity,repeatability,anti-interference ability and subtype detection ability of the newly established assay were evaluated.Results The established SYBR Green real-time fluorescent PCR assay for the detection of five kinds of filarial,including Wuchereria bancrofti,Brugia malayi,Onchocerca volvulus,Loa loa,and Brugia tinori,was specific,without cross-reaction to other related pathogens,including trypanosome.The lower limit of detection of the assay could reach as low as 500 fg per reaction,indicating its high sensitivity.The assay also showed high repeatability.During within batch testing,the cycle threshold(Ct)values were between 32.37 and 33.03,average Ct value was 32.73,SD was 0.20,and the coefficient of variation(CV)was 0.61％.In the interference experiment,the Ct value difference between samples with interference factors and normal samples was 0.79(hemolysis),0.78(lipid blood),0.29(chyluria),and 0.51(decayed mosquito body),respectively,indicating that it can effectively combat the influence of interference factors such as hemeferroheme,lipids,chyluria,and decayed mosquito tissue.Conclusion The established SYBR Green real-time fluorescent PCR assay showed high sensitivity,specificity,repeatability and anti-influence ability,which could provide a rapid method in the detection of filarial.
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