蜜蜂幼虫芽孢杆菌微滴式数字PCR检测方法的建立
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摘要
为建立新型高灵敏度的幼虫芽孢杆菌微滴式数字PCR (droplet digital PCR,ddPCR)检测方法,针对幼虫芽孢杆菌16S rRNA基因设计特异性引物和探针。通过对反应体系优化,建立幼虫芽孢杆菌ddPCR检测方法,并对方法的灵敏度、线性关系、特异性及重复性进行评估。结果:建立的方法与其他6株非幼虫芽孢杆菌无交叉反应,具有良好的特异性;线性关系良好(R~2=0. 991 4);灵敏度为2. 9 copies/μL; ddPCR检测的相对标准偏差(RSD)在0. 93%～5. 27%,重复性好。试验表明本研究建立的ddPCR方法可以对幼虫芽孢杆菌进行绝对定量检测。
This study was to establish a droplet digital PCR (dd PCR) for detection of Legionella pneumophila. Primers and probes were designed for dd PCR according to the 16 S rRNA gene. The dd PCR condition and its reaction system were optimized simultaneously. The dd PCR was evaluated for linearity,specificity,sensitivity and repeatability. The results showed that the high specificity of these primers and probes were evaluated by using 6 non-targeted bacteria. The detection limits of dd PCR were 2. 9 copies/μL. The method had a high degree of linearity (R~2= 0. 991 4). The relative standard deviation (RSD) of dd PCR (0. 93%-5. 27%) was of high repeatability. The dd PCR was of high sensitivity and specificity and might be used to quantitatively detect Paenibacillus larvae.
This study was to establish a droplet digital PCR (dd PCR) for detection of Legionella pneumophila. Primers and probes were designed for dd PCR according to the 16 S rRNA gene. The dd PCR condition and its reaction system were optimized simultaneously. The dd PCR was evaluated for linearity,specificity,sensitivity and repeatability. The results showed that the high specificity of these primers and probes were evaluated by using 6 non-targeted bacteria. The detection limits of dd PCR were 2. 9 copies/μL. The method had a high degree of linearity (R~2= 0. 991 4). The relative standard deviation (RSD) of dd PCR (0. 93%-5. 27%) was of high repeatability. The dd PCR was of high sensitivity and specificity and might be used to quantitatively detect Paenibacillus larvae.
引文
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[4]何晓杰,叶尔保勒,王科珂,等.蜜蜂幼虫芽孢杆菌TaqMan荧光定量PCR检测方法的建立[J].中国预防兽医学报,2017,39(7):569-572.
[5]张其安,王娟,杨少波.蜜蜂细菌性疾病及其防治的研究进展[J].中国蜂业,2011,62:25-30.
[6]Hindson B J,Ness K D,Masquelier D A,et al.High-throughput droplet digital PCR system for absolute quantitation of DNA copy number[J].Anal Chem,2011,83(22):8604-8610.
[7]Anna Kristina Witte,Susanne Fister,Patrick Mester,et al.Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR[J].Anal Bioanal Chem,2016,408:7583-7593.
[8]于纪棉,李如松,倪健波,等.嗜肺军团菌微滴式数字PCR检测方法的建立[J].中国卫生检验杂志,2017,27(9):1233-1236,1239.
[9]郝中香,林华,佘容,等.鲤疱疹病毒2型微滴式数字PCR快速检测方法的建立[J].中国兽医科学,2016,46(2):167-173.
[10]Huang J T,Liu Y J,Wang J,et al.Next generation digital PCRmeasurement of hepatitis B virus copy number in formalin-fixed paraffin-embedded hepatocellular carcinoma tissue[J].Clin Chem,2015,61(1):290-296.
[11]Paola C,Elisabetta G,Noonan DM,et al.A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer[J].BMC Biotechnol,2016,16(6):60.
[12]胡伟,陈荣华,张晨,等.微滴式数字PCR技术用于生物样品种属鉴定和绝对定量[J].法医学杂志,2014,30(5):342-345.
[13]苗丽,张秀平,陈静,等.微滴数字PCR法对肉制品中牛源和猪源成分的定量分析[J].食品工业科技,2016,37(8):187-191.
[14]任怡菲,高琴,邓婷婷,等.基于数字PCR的转基因水稻LL62品系精准定量检测方法建立[J].生物技术通报,2016,32(8):69-76.
[15]OIE.Manual of diagnosis tests and vaccines for terrestrial animals[S].CHAPTER 2.2.2.2017.
[16]Krongdang S,Evans J D,Chen Y et al.Comparative susceptibility and immune responses of Asian and European honey bees to the A-merican foulbrood pathogen,Paenibacillus larvae[J].Insect Sci,2018,doi:10.1111/1744-7917.12593.
[17]李亮,隋志伟,王晶,等.基于数字PCR的单分子DNA定量技术研究进展[J].生物化学与生物物理进展,2012,39(10):1017-1023.
[18]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.
[19]Henrich T J,Gallien S,Li J Z,et al.Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR[J].J Virol Method,2012,186(1):68-72.