卢勇,贾继宗,唐静,叶祥忠
LU Yong,JIA Ji-zong,TANG Jing,YE Xiang-zhong

• 1:北京万泰生物药业股份有限公司
• 2:中国疾病预防控制中心病毒病预防控制所

摘要(Abstract)：

目的比较PCR法、酶法和DNA荧光染色法3种支原体检测方法的灵敏度。方法支原体阳性的BSR细胞培养上清10倍系列稀释后(10-1~10-8),接种至支原体阴性的Vero细胞上,盲传培养5代,每代每个稀释度的样品分别采用PCR法、酶法和DNA荧光染色法检测支原体,并以支原体阴性的Vero细胞作为阴性对照。结果 10倍系列稀释的阳性样品盲传1代,PCR法能检测到10-4,酶法和DNA荧光染色法能检测到10-3;盲传2代,3种方法均能检测到10-4;盲传3代后,3种方法均能检测到10-5,且检测的支原体滴度不随盲传代次的增加而增加。结论待检样本盲传3代后的支原体用3种方法均可检出,检测灵敏度一致。PCR法与酶法检测支原体准确、快速、简便易行,可作为DNA荧光染色法(支原体检测的金标准)的补充手段。
Objective To compare PCR, enzymic method and DNA fluorescent staining for determination of mycoplasmas.Methods Mycoplasma-positive BSR cell culture supernatant was 10-fold serially diluted(10-1 ~ 10-8) and inoculated to mycoplasma-negative Vero cells for five blind passages. The samples of various passages at each dilution were determined for mycoplasma by PCR, enzymic method and DNA fluorescent staining, using mycoplasma-negative Vero cells as control.Results After one blind passage, mycoplasma was detected in the positive samples at a dilution of 10-4by PCR, while in those at a dilution of 10-3by enzymic method and DNA fluorescent method. After two blind passages, mycoplasma was detected in samples at a dilution of 10-4by three methods. However, after three blind passages, mycoplasma was detected in samples at a dilution of 10-5by three methods, of which the titer showed no increase with the increasing passage.Conclusion After the samples were subjected to three blind passages, mycoplasma was detected by each of the three methods with consistent sensitivities. PCR and enzymic method were accurate, rapid and simple for determination of mycoplasma, which might be used as a supplement of DNA fluorescent staining.

关键词(KeyWords)： 支原体;聚合酶链反应;酶法;DNA荧光染色法
Mycoplasma;Polymerase chain reaction;Enzymic method;DNA fluorescent staining

Abstract:

Keywords:

基金项目(Foundation):

作者(Author): 卢勇,贾继宗,唐静,叶祥忠
LU Yong,JIA Ji-zong,TANG Jing,YE Xiang-zhong

DOI: 10.13200/j.cnki.cjb.001091

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