Development and verification of loop-mediated isothermal amplification for detection of 11 common mycoplasmas
10.13200/j.cnki.cjb.003968
- VernacularTitle:11种常见支原体环介导等温扩增检测方法的建立及验证
- Author:
KE Xiaomei
- Publication Type:Journal Article
- From:
Chinese Journal of Biologicals
2023;36(8):980-986
- CountryChina
- Language:Chinese
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Abstract:
Objective To develop and verify a loop-mediated isothermal amplification(LAMP)method for simultaneous detection of multiple mycoplasma contamination.Methods The conserved sequences of 16S rRNA genes of 13 mycoplasma species were aligned by the multiple sequence alignment tool of SnapGene software.Two internal primers FIP and BIP,two external primers F3 and B3 and loop primer LOOP(LF/LB)were designed according to the conserved sequences.The LAMP system was developed as follows:Bst DNA polymerase large segment(8 U/μL),10×ThermoPoly reaction buffer[20 mmol/L Tris-HCl,10 mmol/L KCl,10 mmol/L(H_4)_2SO_4,2 mmol/L MgSO_4 and 0.1%Triton X-100,pH 8.8],MgSO_4(100 mmol/L),dNTP Mix,FIP/BIP Primers,F3/B3 Primers,LOOP Primers,nuclease-free water and template DNA(13 plasmids carrying mycoplasma 16S rRNA).The products were identified by 1%agarose gel electrophoresis.The feasibility,specificity and repeatability of the method were verified,and the detection limit was determined.Results A total of 11 mycoplasma sequences were detected simultaneously by using two sets of LAMP primers screened.There was no nonspecific amplification in other nonspecific template detection,and the primer group had good specificity.The developed method showed good reproducibility and good sensitivity with the minimum detection limit of 30.1 copies/μL,and no false positive or false negative amplification.Conclusion LAMP technology can be used to detect a variety of mycoplasma contamination in cell culture,with good specificity and short time consumption,which can be used as one of the technical platforms for rapid detection of mycoplasma contamination in cell culture.
