Pinheiro de Oliveira Tatiana Flávia, Fonseca Antônio Augusto, Camargos Marcelo Fernandes, de Oliveira Anapolino Macedo, Pinto Cottorello Ana Cláudia, Souza Antonizete Dos Reis, de Almeida Iassudara Garcia, Heinemann Marcos Bryan
Laboratório de Biologia Molecular/Laboratório de Diagnóstico de Doenças Virais, Laboratório Nacional Agropecuário de Minas Gerais, Pedro Leopoldo, Minas Gerais, Brazil.
Biologicals. 2013 Nov;41(6):407-14. doi: 10.1016/j.biologicals.2013.08.005. Epub 2013 Sep 23.
The aim of this study was standardization and application of polymerase chain reaction (PCR) for the detection of contaminants in cell cultures, sera and trypsin. Five PCR protocols were standardized to assess the presence of genetic material from mycoplasma, porcine circovirus 1 (PCV1), bovine leukemia virus (BLV) or bovine viral diarrhea virus (BVDV) in cell culture samples. PCR reactions for the genes GAPDH and beta-actin were used to evaluate the efficiency of nucleic acid extraction. The PCR protocols were applied to 88 cell culture samples from eight laboratories. The tests were also used to assess potential contamination in 10 trypsin samples and 13 fetal calf serum samples from different lots from five of the laboratories. The results showed the occurrence of the following as DNA cell culture contaminants: 34.1% for mycoplasma, 35.2% for PCV1, 23.9% for BVDV RNA and 2.3% for BLV. In fetal calf sera and trypsin samples BVDV RNA and PCV1 DNA was detected. The results demonstrated that cell culture, sera and trypsin used by different laboratories show a high rate of contaminants. The results highlight the need for monitoring cell cultures and controlling for biological contaminants in laboratories and cell banks working with these materials.
本研究的目的是对用于检测细胞培养物、血清和胰蛋白酶中污染物的聚合酶链反应（PCR）进行标准化及应用。标准化了五种PCR方案，以评估细胞培养物样本中支原体、猪圆环病毒1型（PCV1）、牛白血病病毒（BLV）或牛病毒性腹泻病毒（BVDV）的遗传物质的存在情况。使用针对甘油醛-3-磷酸脱氢酶（GAPDH）基因和β-肌动蛋白基因的PCR反应来评估核酸提取效率。将这些PCR方案应用于来自八个实验室的88个细胞培养物样本。这些检测还用于评估来自五个实验室不同批次的10个胰蛋白酶样本和13个胎牛血清样本中的潜在污染情况。结果显示，作为DNA细胞培养污染物出现的情况如下：支原体为34.1%，PCV1为35.2%，BVDV RNA为23.9%，BLV为2.3%。在胎牛血清和胰蛋白酶样本中检测到了BVDV RNA和PCV1 DNA。结果表明，不同实验室使用的细胞培养物、血清和胰蛋白酶显示出较高的污染物发生率。这些结果突出了在使用这些材料的实验室和细胞库中监测细胞培养物和控制生物污染物的必要性。
