[Establishment of LAMP Detection for Toxoplasma gondii Based on the 529 bp Repeat Sequence].

Abstract

Objective: To establish the loop mediated isothermal amplification（LAMP） technique for determining Toxoplama gondii.

Methods: The primers for LAMP of the conserved 529 bp sequence was designed by the Primer Explorer 4.0 software. The LAMP reaction was made on the constructed pMD-19T-529 bp recombinant plasmid as a template, and was optimized in loop primer, concentrations of betaine and MgSO4, and reaction temperature. The optimized LAMP and PCR were performed in ultra-pure water, on pig genome, Cryptosporidium parvum genome, Isospora suis genome, and pMD-19T-529 bp, respectively, to test the sensitivity of LAMP. The pMD-19T-529 bp was serially diluted to 109-100 copies/ml to test the specificity of LAMP.

Results: LAMP using the designed primers amplified the 529 bp fragment of T. gondii. The optimized LAMP system had a total reaction volume of 25 μl, with the optimal concentrations of betaine and MgSO4 being 0.4 mol/L and 6 mmol/L, respectively. The amplification efficiency of 30 min reaction in the presence of loop primer was comparable to that of 60 min reaction without loop primer, which indicated that addition of loop primer shortened the reaction time by 30 min. The optimal reaction condition was 63 ℃ 30 min, 80 ℃ 3 min. The established LAMP method specifically amplifed the 529 bp fragment of T. gondii, and its efficiency was 10 times of PCR（detection threshold 103 copies/ml vs. 104 copies/ml）.

Conclusion: The established LAMP for detecting Toxoplama gondii 529 bp repeat sequence shows high specificity and sensitivity.