ACT1-qPCR定量分析弓形虫内参引物的筛选与鉴定

中国寄生虫学与寄生虫病杂志 ›› 2018, Vol. 36 ›› Issue (4): 375-380.

ACT1-qPCR定量分析弓形虫内参引物的筛选与鉴定

郭海婷^1,², 陈志宝¹, 李中原^2,^3,^*()

1 黑龙江八一农垦大学生命科学技术学院,大庆 163319
2 中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃省动物寄生虫病重点实验室,兰州 730046
3 安徽农业大学动物科技学院,合肥 230036

收稿日期:2018-02-19 出版日期:2018-08-30 发布日期:2018-09-06
通讯作者: 李中原
基金资助:
黑龙江八一农垦大学研究生创新科研项目（No. YJSCX2017-Y71）;家畜疫病病原生物学国家重点实验室开放基金（No. SKLVEB2016KFKT015）

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

Hai-ting GUO^1,², Zhi-bao CHEN¹, Zhong-yuan LI^2,^3,^*()

1 College of Biological Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China
2 State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
3 College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China

Received:2018-02-19 Online:2018-08-30 Published:2018-09-06
Contact: Zhong-yuan LI
Supported by:
Supported by the Graduate Innovation Research Project of Heilongjiang Bayi Agricultural University(No. YJSCX2017-Y71), and the Open Fund of the State Key Laboratory of Veterinary Etiological Biology(No. SKLVEB2016KFKT015)

摘要/Abstract

摘要：

目的运用PCR对刚地弓形虫（Toxoplasma gondii）RH、PRU、VEG株速殖子和HFF、Vero细胞系以及SPF级昆明鼠脑组织所得DNA样品进行特异性扩增,鉴定并筛选出适合用作弓形虫实时荧光定量PCR（qPCR）分析的内参引物。方法体外培养并无菌收集HFF、Vero细胞系及弓形虫RH、PRU、VEG株速殖子,SPF级昆明鼠经颈椎脱臼处死无菌采集脑组织,并提取基因组DNA。合成弓形虫MIC6基因（ToxoDB：TGME49_218520）特异性引物P1/P2,经PCR扩增鉴定所得样品是否含有弓形虫DNA成分,设空白对照。依照ToxoDB数据库弓形虫ACT1基因参考序列（ToxoDB：TGME49_209030）设计引物,经NCBI Primer-BLAST比对分析后进行合成特异性引物P3/P4、P5/P6、P7/P8、P9/P10和P11/P12,预期片段大小均为121 bp。以弓形虫RH DNA为模板,采用梯度PCR优化ACT1基因内参引物的退火温度。运用已优化条件对待检DNA样品进行PCR特异性扩增和琼脂糖凝胶电泳分析,鉴定出适合用于弓形虫qPCR定量分析的内参引物。结果 PCR扩增弓形虫RH、PRU和VEG株DNA MIC6基因时均出现一条长度约为1 050 bp的条带,且空白对照和其他样品无此条带,与预期结果一致,表明所得DNA样品可用。优化退火温度时发现,除P7/P8外其他引物均不受退火温度影响,56 ℃适合用于PCR或qPCR扩增ACT1基因目的片段的退火温度。以56 ℃为退火温度对待检DNA进行检测时发现,扩增弓形虫阳性样品时所有引物均能产生1条大小约为121 bp的条带,空白对照无此条带,与预期结果一致。此外,引物P7/P8、P9/P10扩增Vero细胞系和引物P3/P4、P5/P6、P9/P10扩增昆明鼠脑组织DNA样品时均有杂带出现,且有杂带大小约为121 bp;引物P11/P12扩增弓形虫阴性DNA样品时均无杂带出现。结论引物P11（5′-TCGGTGACGAAGCCCAAA-3′）和P12（5′-AGTTCGTTGTAGAAGGTGTGA-3′）适合分别用作弓形虫qPCR定量分析时扩增ACT1靶基因的上游和下游引物。

关键词: 刚地弓形虫, ACT1, qPCR, 内参引物, 鉴定

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

中图分类号:

R382.5

郭海婷, 陈志宝, 李中原. ACT1-qPCR定量分析弓形虫内参引物的筛选与鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(4): 375-380.

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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