基于聚合酶链式反应（PCR）的支原体检测方法验证概念的介绍

Introduction of a validation concept for a PCR-based Mycoplasma detection assay.

作者信息

Bruchmüller I, Pirkl E, Herrmann R, Stoermer M, Eichler H, Klüter H, Bugert P

机构信息

Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg-Hessen, University of Heidelberg, Faculty of Clinical Medicine Mannheim, Mannheim, Germany.

出版信息

Cytotherapy. 2006;8(1):62-9. doi: 10.1080/14653240500518413.

DOI:10.1080/14653240500518413

PMID:16627346

Abstract

BACKGROUND

Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept.

METHODS

The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values.

RESULTS

The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale.

DISCUSSION

The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.

摘要

背景

支原体污染是细胞培养中最常见的问题之一。为了满足细胞系和治疗产品支原体检测的法律要求（欧洲药典和美国食品药品监督管理局），我们开发了一种基于聚合酶链反应（PCR）的方法来检测支原体，并引入了验证概念。

方法

该PCR检测法特异性扩增编码16S核糖体DNA（rDNA）的基因的一段280碱基对（bp）的DNA片段。同时扩增包含引物结合位点的人工寡核苷酸可控制PCR的效率。使用两种支原体参考菌株口腔支原体和肺炎支原体对PCR检测法进行验证。验证概念包括：（i）在含有细菌代谢指示剂的培养基中培养口腔支原体和肺炎支原体；（ii）在重复稀释实验中测定变色单位（CCU）；（iii）将PCR结果与CCU值进行关联。