為建立一種靈敏、可快速檢測(cè)豬圓環(huán)病毒4型（PCV4）的SYBR Green I實(shí)時(shí)熒光定量PCR方法，根據(jù)PCV4 Rep基因的保守區(qū)域設(shè)計(jì)引物，建立針對(duì)PCV4的SYBR Green I實(shí)時(shí)熒光定量PCR檢測(cè)方法，并對(duì)該方法的特異性、靈敏性和重復(fù)性進(jìn)行評(píng)價(jià)。結(jié)果顯示，該檢測(cè)方法的Ct值與標(biāo)準(zhǔn)品在5.64×102~5.64×109 copies/μL范圍內(nèi)呈良好的線性關(guān)系，R2為0.999，斜率為-3.638，檢測(cè)下限為5.64×102 copies/μL，且無非特異性擴(kuò)增。利用該方法對(duì)56份臨床樣品進(jìn)行檢測(cè)，發(fā)現(xiàn)PCV4核酸陽性率為3.57%，比普通PCR更靈敏可靠。結(jié)果表明，本試驗(yàn)建立的SYBR Green I實(shí)時(shí)熒光定量PCR方法可用于PCV4的快速診斷。
Establishment and Application of SYBR Green I Real-time PCR for Detection of PCV4
In order to rapidly and sensitively detect porcine circovirus type 4（PCV4），the SYBR Green I real-time quantitative PCR assay was established based on the primers that were designed according to the conserved region of PCV4 Rep gene. Then its specificity，sensitivity and repeatability were evaluated. The results showed that its Ct value remained a good linear relationship with the standard materials within the range of 5.64×109~5.64×102 copies/μL，specifically，R2 was 0.999，the slope was -3.638，and the detection limit was 5.64×102 copies/μL without non-specific amplification. 56 clinical samples were tested by the method，it was found that the nucleic acid positive rate of PCV4 was 3.57%，which was more sensitive and reliable than that of conventional PCR. Therefore，the established SYBR Green I real-time PCR method could achieve the rapid detection of PCV4.
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