Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

Kazemiha, Vahid Molla; Bonakdar, Shahin; Amanzadeh, Amir; Azari, Shahram; Memarnejadian, Arash; Shahbazi, Shirin; Shokrgozar, Mohammad Ali; Mahdian, Reza

doi:10.1007/s10616-015-9862-0

Cited by 12 publications

(11 citation statements)

References 49 publications

(59 reference statements)

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“…In 1956, Johns Hopkins first reported mycoplasma contamination of HeLa cells cultured 40. Mycoplasma can disturb cultured cell biological and biopharmaceutical studies, and infection rates can range from 15 to 100% 41. In our study, ZJU-0826, and -1125 were free of mycoplasma contamination as determined by PCR.…”

Section: Discussionmentioning

confidence: 45%

Establishment and Characterization of Two Novel Cholangiocarcinoma Cell Lines

et al. 2019

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BackgroundCholangiocarcinoma (CCA) is a rare, aggressive and highly lethal tumor. The disease is very difficult to diagnose and multi-modality treatments are ineffective. To improve our understanding of the biological characteristics of CCA, and facilitate the identification of valid treatments, an in-depth characterization of two novel Chinese patient-derived primary CCA cell lines was performed.MethodsTwo CCA cell lines were developed and labelled ZJU-0826 and -1125. The two cell lines were characterized with respect to phenotypic, molecular, biomarker, functional and histological properties.ResultsTwo novel cell lines were cultured for 2 years, and maintained for more than 100 passages. They retained their typical biliary epithelial morphology and ultrastructure. The population doubling times of ZJU-0826, and -1125 were 63.84 h and 44.73 h, respectively. The cells exhibited near-triploid karyotypes with complex structural aberrations. ZJU-1125 cells had mutations in TP53 exons. Short tandem repeats genotyping confirmed the human origin and difference between lines. An immunophenotype analysis showed that ZJU-0826 is positive for CD44, CD29, Pdx1, CD236, FoxA1, FoxA2, and Nanog, and ZJU-1125 positive for CD44, CD29, CD133, Pdx1, FoxA1, FoxA2, and Nanog. ZJU-1125 had greater invasion ability in vitro and tumorigenicity than those of ZJU-0826.ConclusionsOur results confirm the validity of the ZJU-0826 and -1125 as representative models for the elucidation of the molecular pathogenesis of perihilar CCA, and intrahepatic CCA in both in vitro and in vivo studies, respectively.Electronic supplementary materialThe online version of this article (10.1245/s10434-019-07649-5) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 45%

Establishment and Characterization of Two Novel Cholangiocarcinoma Cell Lines

et al. 2019

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“…*P < 0.05, **P < 0.01 production relies on the hydrolysis of urea [16]. Finally, the sensitivity of biochemical detection has been shown to be lower than that for PCR or qPCR methods [17,18].…”

Section: Discussionmentioning

confidence: 99%

Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

et al. 2019

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Objective: We aim to directly detect Mycoplasma DNA in a U937 suspension cell culture without using DNA purification. In order to make Mycoplasma contamination monitoring easier, we optimized a commercially available quantitative PCR (qPCR)-based detection kit. We compared the sensitivity of direct qPCR against qPCR with a purified DNA template. Results: Our findings indicate that qPCR worked optimally with a 6 μl sample volume and a 52 °C annealing-extension temperature. We were able to decrease the annealing-extension step time from 60 to 20 s without any major decrease in reaction sensitivity. The total cycle time of optimized direct qPCR was 65 min. The optimized qPCR protocol was used to detect Mycoplasma DNA before and after DNA purification. Our findings indicate that direct qPCR had a higher sensitivity than regular qPCR. Ct levels produced by direct qPCR with 6 μl templates were almost identical to Ct levels produced by regular qPCR with DNA purified from a 60 μl cell culture sample (23.42 vs 23.49 average Ct levels, respectively). The optimized direct qPCR protocol was successfully applied to monitor the elimination of Mycoplasma contamination from U937 cell cultures.

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“…Lastly, the sensitivity of the biochemical detection was shown to be lower than that for PCR or qPCR methods [17,18]. There is a variety of kits on offer based on regular PCR, followed by gel electrophoresis.…”

Section: Discussionmentioning

confidence: 99%

Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

Baaity

Breunig

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et al. 2019

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Objective In order to make the Mycoplasma contamination monitoring task easier, we optimized a commercially available quantitative PCR (qPCR)-based detection kit to detect Mycoplasma DNA without DNA purification in a U937 suspension cell culture. We compared the sensitivities of the direct qPCR and the qPCR with the purified DNA template. Results Our findings indicate that qPCR worked optimally with a 6 ml sample volume and a 52 o C annealing-extension temperature. We were able to decrease the annealing-extension step time from 60 sec to 20 sec without any major decrease in the reaction sensitivity. The total cycle time of the optimized direct qPCR was 65 minutes. The optimized qPCR protocol was used to detect Mycoplasma DNA directly and after DNA purification. Our findings indicate that the direct qPCR had a higher sensitivity compared to the regular qPCR. The Ct levels produced by the direct qPCR with 6 ml templates were almost identical to the Ct levels produced by the regular qPCR with DNA purified from a 60 ml cell culture sample (23.42 vs 23.49 average Ct levels respectively). The optimized direct qPCR protocol was successfully applied to monitor the elimination of Mycoplasma contamination from the U937 cell cultures.

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Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

Cited by 12 publications

References 49 publications

Establishment and Characterization of Two Novel Cholangiocarcinoma Cell Lines

Establishment and Characterization of Two Novel Cholangiocarcinoma Cell Lines

Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

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