Screening and identification of primers for internal control used in ACT1-qPCR analysis of Toxoplasma gondii

Abstract

Abstract:

Objective PCR was performed on DNA extracted from tachyzoites of Toxoplasma gondii RH, PRU and VEG strains, HFF and Vero cell lines, and brain tissues of SPF-grade Kunming mice, in order to identify primers for internal control used to quantify Toxoplasma gondii by quantitative PCR (qPCR). Methods Genomic DNA was extracted from in vitro cultured HFF and Vero cells and tachyzoites of T. gondii RH, PRU and VEG strains. A SPF-grade Kunming mouse was sacrificed by cervical dislocation and the brain tissue was isolated for DNA extraction. The P1/P2 primer specific for T. gondii MIC6 gene (ToxoDB: TGME49_218520) was synthesized according to previous reports, and used for PCR amplification using the extracted DNA samples as template. The presence of T. gondii DNA was examined based on the PCR product bands. PCR with omission of DNA template was used as a blank control. Primers for T. gondii ACT1 gene (ToxoDB: TGME49_209030) were designed, underwent NCBI Primer-BLAST alignment, and synthesized as P3/P4, P5/P6, P7/P8, P9/P10 and P11/P12, with an expected band size of 121 bp. Using T. gondii RH DNA as the template, the annealing temperatures of the primers were optimized by gradient PCR. The optimized PCR conditions were used to amplify the DNA samples, and agarose gel electrophoresis was performed to identify the suitable internal control primers for qPCR of T. gondii. Results As expected, a single band of ~1 050 bp was seen after PCR amplification of T. gondii MIC6 gene using DNA samples from T. gondii RH, PRU and VEG strains, while no band was seen in the blank control group and on other samples, suggesting that the obtained DNA samples can be used in the following examinations. Optimization of PCR annealing temperatures revealed that all primer candidates except the P7/P8 performed comparably at different annealing temperatures, and 56 ℃ was selected as the annealing temperature to amplify ACT1 gene by PCR and qPCR. As expected, using this temperature, all primers resulted in a band of ~121 bp, while no band was seen in the blank control group. In addition, non-target bands were seen after amplification of Vero DNA using primers P7/P8 and P9/P10, and after amplification of Kunming mouse brain DNA using primers P3/P4, P5/P6 and P9/P10, some of which were ~121 bp. Notably, primer P11/P12 was specific and no non-target band was seen after amplification of the Toxoplasma negative samples. Conclusion Primers P11 (5′-TCGGTGACGAAGCCCAAA-3′)/P12 (5′-AGTTCGTTGTAGAAGGTGTGA-3′) are suitable as forward and reverse primers used in qPCR analysis of T. gondii ACT1 gene.

Key words: Toxoplasma gondii, ACT1, qPCR, Internal control primer, Identification

CLC Number:

R382.5

Hai-ting GUO, Zhi-bao CHEN, Zhong-yuan LI. Screening and identification of primers for internal control used in ACT1-qPCR analysis of Toxoplasma gondii[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2018, 36(4): 375-380.

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