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Identification and verification of rodent cell lines by polymerase chain reaction.

ABSTRACT: Cell lines represent valuable tools for basic research and diagnostic applications as well as for the production of biological products such as antibodies or vaccines. For all cell culturists, a well-identified origin of their cell lines as well as the periodic re-examination of their identity should be a basic requirement. We established a simple polymerase chain reaction (PCR) to verify or identify rodent and human cell lines. Since mouse-, rat-, Chinese hamster- and Syrian hamster-derived cell lines represent the most frequently used rodent cell lines, our investigations were focused on these species. Our assay used oligonucleotide primers annealing to sequences within the beta-actin and the beta-globin gene and to repetitive DNA. Primers were designed mostly from intron sequences of the genes aiming to amplify only one specific DNA segment and thus enabling to exclude easily false DNA. More than 130 cells lines originating from the five species were analyzed in that study. Our PCR revealed specific profiles for all species investigated. No further methods like DNA sequencing or fragment length polymorphism analysis were needed to differentiate these species. The results introduce our PCR-assay as a rapid, specific and routinely feasible tool in order to identify or distinguish rodent cell lines from each other and from human cell lines.

SUBMITTER: Steube KG

PROVIDER: S-EPMC2151966 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Identification and verification of rodent cell lines by polymerase chain reaction.

Steube Klaus G KG Koelz Anne-Leena AL Drexler Hans G HG

Cytotechnology 20071031 1

Cell lines represent valuable tools for basic research and diagnostic applications as well as for the production of biological products such as antibodies or vaccines. For all cell culturists, a well-identified origin of their cell lines as well as the periodic re-examination of their identity should be a basic requirement. We established a simple polymerase chain reaction (PCR) to verify or identify rodent and human cell lines. Since mouse-, rat-, Chinese hamster- and Syrian hamster-derived cel ...[more]

PMID: 19002841

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Project description:Background and aimDifferent polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and methodsOne hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.ResultsTaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.ConclusionA cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

Dataset Information

Identification and verification of rodent cell lines by polymerase chain reaction.

Publications

Identification and verification of rodent cell lines by polymerase chain reaction.

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