人博卡病毒微滴数字PCR定量方法的建立
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摘要
为准确、快速地从临床鼻咽拭子样本中筛选出人博卡病毒(HBoV)感染的阳性样本,以HBoV-sh9基因的保守区序列为检测靶标合成引物和探针,建立了微滴数字PCR(dd PCR)检测HBoV的方法.结果表明:HBoV dd PCR方法具有较高的特异性,在(0.5~6.8)×10~3copies/μL HBoV DNA浓度范围内,具有良好的线性和精确度,定量限低至0.5 copies/μL.182份临床样本检测结果显示:HBoV感染的阳性率为8.24%.此建立的HBoV dd PCR方法在定量限、准确性和重复性上较常规PCR优势明显,不受样品基质和扩增效率的影响.
In order to accurately and quickly detect positive specimens of human bocavirus( HBoV) infection from clinical nasopharyngeal swab samples,primers and probes were synthesized by using the conserved region sequence of HBoV-sh9 gene as the target and the method of droplet digital polymerase chain reaction( dd PCR) for HBoV detection was established. The results showed that dd PCR method had high specificity,good linearity and precision within the HBoV DNA range of( 0.5 ~ 6.8) × 10~3 copies/μL,and the detection limit was as low as 0. 5 copies/μL. 182 clinical samples were tested and a positive HBoV infection rate of 8. 24% was found. This HBoV dd PCR method had obvious advantages in quantitative limit,accuracy and repeatability compared with the conventional PCR and was not affected by sample matrix and amplification efficiency.
In order to accurately and quickly detect positive specimens of human bocavirus( HBoV) infection from clinical nasopharyngeal swab samples,primers and probes were synthesized by using the conserved region sequence of HBoV-sh9 gene as the target and the method of droplet digital polymerase chain reaction( dd PCR) for HBoV detection was established. The results showed that dd PCR method had high specificity,good linearity and precision within the HBoV DNA range of( 0.5 ~ 6.8) × 10~3 copies/μL,and the detection limit was as low as 0. 5 copies/μL. 182 clinical samples were tested and a positive HBoV infection rate of 8. 24% was found. This HBoV dd PCR method had obvious advantages in quantitative limit,accuracy and repeatability compared with the conventional PCR and was not affected by sample matrix and amplification efficiency.
引文
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[2]YeilbaO,Klhtlr H S,Talip P M,et al.Very rare and life-threatening complications of bocavirus bronchiolitis:pneumomediastinum and bilateral pneumothorax[J].Mikrobiyol Bul,2016,50(1):159-164.
[3]Lu X,Chittaganpitch M,Olsen S J,et al.Real-time PCR assays for detection of bocavirus in human specimens[J].J Clin Microbiol,2006,44(9):3231-3235.
[4]Choi J H,Chung Y S,Kim K S,et al.Development of real-time PCR assays for detection and quantification of human bocavirus[J].J Clin Virol,2008,42(3):249-253.
[5]Lu X,Chittaganpitch M,Olsen S J,et al.Real-time PCR assays for detection of bocavirus in human specimens[J].J Clin Microbiol,2006,44(9):3231-3235.
[6]Endo R,Ishiguro N,Kikuta H,et al.Seroepidemiology of human bocavirus in Hokkaido prefecture,Japan[J].J Clin Microbiol,2007,45(10):3218-3223.
[7]Vogelstein B,Kinzler K W.Digita PCR[J].PNAS,1999,96(16):9236-9241.
[8]陈莹,张丹,李京京,等.人类博卡病毒结构蛋白基因VP2的克隆与表达[J].化学与生物工程,2010,27(3):67-69.
[9]刘钊,马年,钟研,等.大黄素体外抗柯奇病毒B4的实验研究[J].中南民族大学学报(自然科学版),2015,34(1):56-61.
[10]邓幼平,刘颖娟,王笑臣,等.Taqman PCR检测人博卡病毒的阳性率[J].海南医学院学报,2014,20(3):289-292.
[11]何霞,曹开源.人博卡病毒及其在中国的流行现状[J].病毒学报,2013(1):56-64.
[12]林峰,滕灵方,郑美云,等.利用人支气管上皮细胞系分离和培养人博卡病毒[J].中华实验和临床病毒学杂志,2009,23(6):437-439.
[13]Dijkman R,Koekkoek SM,Molenkamp R,et al.Human Bocavirus can be cultured in differentiated human airway epithelial cells[J].J Virol.2009,83(15):7739-7748.
[14]Zheng L S,Yuan X H,Xie Z P,et al.Human bocavirus infection in young children with acute respiratory tract infection in Lanzhou,China[J].J Med Virol,2010,82(2):282-288.
[15]Bonvicini F,Manaresi E,Gentilomi G A,et al.Evidence of human bocavirus viremia in healthy blood donors[J].Diagn Microbiol Infect Dis,2011(4),71:460-462.
[16]张丽,刘丽丽,梁晓声,等.定时定量PCR鉴定转基因作物纯合体[J].中南民族大学学报(自然科学版),2015,34(1):43-46.
[17]Sanders R,Huggett J F,Bushell C A,et al.Evaluation of digital PCR for absolute DNA quantification[J].Anal Chem,2011,83(17):6474-6484.
[18]White R A,Quake S R,Curr K.Digital PCR provides absolute quantitation of viral load for an occult RNA virus[J].J Virol Methods,2012,179(1):45-50.
[19]Strain M C,Lada S M,Luong T,et al.Highly precise measurement of HIV DNA by droplet digital PCR[J].Plo S one,2013,8(4):e55943.