为了建立绿脓杆菌的SYBR Green I实时定量PCR检测方法,以便更加快捷、方便的检测绿脓杆菌,根据16S rDNA高度保守的特性,在绿脓杆菌16S rDNA保守区设计1对引物,利用普通PCR技术扩增出绿脓杆菌16S rDNA保守区277 bp的片段,并克隆到pMD-18 T载体上,纯化的质粒作为模板进行SYBR Green Ⅰ荧光定量PCR扩增并制作标准曲线,建立了绿脓杆菌的荧光定量PCR检测方法。并对方法的灵敏性、特异性、重复性进行评价,又进一步在临床实践中进行检验。结果显示,所建立的方法对标准样品的最小检出浓度为28拷贝/μL。并且特异性检验结果显示与常见的菌群没有交叉反应,重复性良好。在临床中检测疑似绿脓杆菌感染病料55份,用所建立的荧光定量方法检测出阳性病料40份,而普通的PCR方法检测出阳性病料32份。表明建立的实时荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点,可以在临床中用于绿脓杆菌的检测。
宋月
,
程琨
,
梁秀丽
,
王亚宾
,
付彤
,
韩立强
,
魏战勇
. 绿脓杆菌SYBR GreenI实时定量PCR检测方法的建立及初步应用[J]. 华北农学报, 2014
, 29(3)
: 59
-63
.
DOI: 10.7668/hbnxb.2014.03.012
To development the SYBR Green I real-time quantitative PCR assay for detection of Pseudomonas aeruginosa quicker and more convenient.According to the characteristics of highly conserved of Pseudomonas aeruginosa 16S rDNA, we designed a pair of primers to establish the quantitative PCR methods.We got a 277 bp region of the Pseudomonas aeruginosa 16S rDNA was amplified using normal PCR.Then sub-cloned to pMD-18 T vector and acquired the recombinant plasmid, which served as template to conduct the standards curve of the SYBR Green I real-time PCR.And the sensitivity, specificity and reproducibility of our method were evaluated, and further testing was done in clinical practice.The sensitivity analysis showed that the developed SYBR Green I real-time PCR could detect 28 coppies/μL.Specificity testing results showed that it has no cross-react with common environmental bacteria.Then the established method was used to detect the clinical samples.The results showed that 40 positive samples out of 55 suspicious positive samples could be observed by real-time PCR and 32 positive samples could be detected by normal PCR.These results indicated that the SYBR Green I real-time PCR we established in present study showed the characteristics of sensitivity and specificity, and could be used in clinical diagnosis and epidemiological investigation for Pseudomonas aeruginosa.
[1] 王 斌,李旭廷,黄 伟,等.急性临床型奶牛乳房炎病原菌的分离鉴定与药敏试验[J].中国奶牛,2012(5):37-39.
[2] 韦志锋,李智红,陶 立,等.山羊支原体和绿脓杆菌混合感染的诊治[J].中国畜禽种业,2011,7(3):112-113.
[3] 李翠蓉.初生雏鸭绿脓杆菌感染[J].中国畜牧兽医,2010,37(12):221-223.
[4] 靳双星,陈理盾,段继永.雏鸡绿脓杆菌的分离鉴定及药敏试验[J].中国畜牧兽医,2011,38(7):230-232.
[5] 陈红梅,程龙飞,万春和,等.死亡番鸭胚中绿脓杆菌的分离鉴定及药敏试验[J].中国兽医杂志,2012,48(12):40-42.
[6] 吴异健.乳鸽绿脓杆菌病的诊疗报告[J].福建畜牧兽医,2010,32(1):57-58.
[7] 杨培培,王 颖,胡继明,等.水貂铜绿假单胞菌鞭毛分型及分离株 flic 基因的克隆与序列分析[J].中国预防兽医学报,2012,34(9):749-751.
[8] 鲍国连,佟承刚,韦 强,等.家兔绿脓杆菌病原特性和致病作用的研究[J].中国养兔,2001(5):3-4.
[9] 蔡双福,周芳梅,黄楚妮.水样中绿脓杆菌产色素的初步研究[J].现代食品科技,2011,27(6):640-642.
[10] 吴晨璐,施春雷,周 敏,等.食源性肠球菌荧光定量PCR检测方法的建立与评价[J].食品与生物技术学报,2011,30(5):779-787.
[11] Shin S W,Cha S B,Lee W J,et al.Application of SYBR Green real-time PCR assay for the specific detection of Salmonella spp[J].Korean Journal of Veterinary Research,2013,53(1):25-28.
[12] 李明凤,魏战勇,王学斌,等.猪细小病毒SYBR Green Ⅰ实时定量PCR检测方法的建立[J].浙江农业学报,2009,21(3):220-224.
[13] 胡兴娟,王淑娜,周向阳,等.水产品中的副溶血弧菌和霍乱弧菌双重荧光定量PCR快速检测方法的建立[J].河南农业科学,2010(9):121-124.
[14] 赵绪永,宁豫昌,赵 丽,等.多重实时定量PCR快速检测PRRSV、CSFV和PCV2混合感染方法的建立[J].河南农业科学,2013,42(2):123-127.
[15] 李碧侠,赵 芳,任守文,等.猪 SIRT1 基因荧光定量PCR检测方法的建立[J].华北农学报,2011,26(增刊):44-46.
[16] 张 莉,王英珍,鄢明华,等.羊流产衣原体的PCR检测方法研究[J].天津农业科学,2009,15(2):14-16.
[17] 樊振华,王娟萍,孟 帆,等.猪细小病毒和猪圆环病毒2型多重PCR检测方法的建立与应用[J].山西农业科学,2012,40(10):1102-1106.
[18] 高 旭,鲁 承,杜秋明.鹅细小病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立及应用[J].河南农业科学,2010(12):121-124.