Detection of contaminants in cell cultures, sera and trypsin.
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.
Laboratório de Biologia Molecular/Laboratório de Diagnóstico de Doenças Virais, Laboratório Nacional Agropecuário de Minas Gerais, Pedro Leopoldo, Minas Gerais, Brazil. Electronic address: oliveiratfp@yahoo.com.br.
Tatiana Flávia
TF
Pinheiro de Oliveira
Antônio Augusto
AA
Fonseca
Marcelo Fernandes
MF
Camargos
Anapolino Macedo
AM
de Oliveira
Ana Cláudia
AC
Pinto Cottorello
Antonizete Dos Reis
Ados R
Souza
Iassudara Garcia
IG
de Almeida
Marcos Bryan
MB
Heinemann
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D017925:Circovirus / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D030241:Diarrhea Virus 1, Bovine Viral / Q000235:genetics; D004340:Drug Contamination; D006801:Humans; D007753:Laboratories / Q000592:standards / Q000706:statistics & numerical data; D001909:Leukemia Virus, Bovine / Q000235:genetics; D009174:Mycoplasma / Q000235:genetics; D009175:Mycoplasma Infections / Q000097:blood / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D044967:Serum / Q000382:microbiology* / Q000821:virology*; D013552:Swine; D014357:Trypsin / Q000032:analysis*; D014777:Virus Diseases / Q000097:blood / Q000821:virology
D004269:DNA, Bacterial; D004279:DNA, Viral; D012367:RNA, Viral; D014357:Trypsin
Cell cultures; Mollicutes; PCR; Sera; Trypsin; Viral contaminants