实验动物弓形虫荧光定量 PCR 检测方法的建立及应用

doi:10.3969/j.issn.1006-6179.2024.04.002

实验动物科学 ›› 2024, Vol. 41 ›› Issue (4): 8-14.DOI: 10.3969/j.issn.1006-6179.2024.04.002

实验动物弓形虫荧光定量 PCR 检测方法的建立及应用

(中国农业科学院哈尔滨兽医研究所,,动物疫病防控全国重点实验室,黑龙江省实验动物与
比较医学重点实验室,国家禽类实验动物资源库,哈尔滨 150069)

收稿日期:2023-01-07 出版日期:2024-08-28 发布日期:2024-08-28
通讯作者: 夏长友( 1972—) ,男,研究员,研究方向为实验动物学,E-mail: xiachangyou@ caas. cn
作者简介:罗霆宇( 1998—) ,男,硕士研究生,研究方向为实验动物质量控制,E-mail: 827731803@ qq. com。
基金资助:
国家重点研发计划项目( 2021YFF0703000) ;国家生猪技术创新中心先导科技项目( NCTIP-XD1C09) ;中央级公益性科研院所

基本科研业务费专项( 1610302022018) ;兽医生物技术国家重点实验室课题( SKLVBP202120,SKLVBP202101)

Establishment and Application of Fluorescent Quantitative PCR Assay for

Laboratory Animal Toxoplasma gondii

#br#

( State Key Laboratory for Animal Disease Control and Prevention, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, National Poultry Laboratory Animal Resource Center, Harbin Veterinary Research Institute,

Chinese Academy of Agricultural Sciences, Harbin 150069,China)

Received:2023-01-07 Online:2024-08-28 Published:2024-08-28

摘要/Abstract

摘要： :目的建立实验动物弓形虫 TaqMan 探针实时荧光定量 PCR 检测方法,并对其进行初步应用。方法通过
比对美国国家生物技术信息中心(NCBI)发表的弓形虫各毒株序列,选取其 529 bp 重复序列保守区域设计引物探
针,建立弓形虫的荧光定量 PCR 方法。随后对方法的特异性和敏感性进行检验;取浓度为 106~ 102copies/ μL 10
倍系列稀释的质粒标准品,每个浓度做 3 个平行,重复检测 3 次,计算组内和组间变异系数,评估方法的重复性和
稳定性;用建立的荧光定量 PCR 方法及国标推荐的 PCR 方法检测 14 份犬组织样品和 66 份猪全血样品,以评估此
方法在实际应用中的检测能力。结果建立的弓形虫荧光定量 PCR 方法在 108~ 101copies/ μL 范围 Ct 值与质粒
浓度呈线性关系,标准曲线 Slope 为-3. 285,R2 值为 1,扩增效率为 101. 663%。可检测到的弓形虫最低浓度为 10
1copies/ μL;与其他 14 种实验猪常见病原不发生交叉反应;重复检测 106~ 102copies/ μL 质粒标准品,组内变异系数
小于 1. 21%,组间变异系数小于 0. 62%。用建立的弓形虫荧光定量 PCR 方法和国标 PCR 方法对 14 份犬组织样品
和 66 份猪全血样品进行检测,结果显示,弓形虫荧光定量 PCR 方法和国标 PCR 方法检测的阳性率分别为 10%(8 /
80)和 7. 5%(6 / 80) ,阳性符合率达到 100%。结论建立的弓形虫荧光定量 PCR 方法可以有效地检测弓形虫,为
实验动物弓形虫日常监测提供良好的方法。

关键词:

弓形虫, 荧光定量 PCR, 应用

Abstract: To establish and apply TaqMan probe real-time fluorescent quantitative PCR
method for laboratory animal Toxoplasma gondii. Method By comparing the sequences of various strains
of Toxoplasma gondii published by NCBI, the conservative region of 529 bp repetitive sequence was
selected to design primers and probe, and a fluorescence quantitative PCR method was established.
Subsequently, the specificity and sensitivity of the method was confirmed. Plasmid standards were

obtained from 106 to 102copies/ μL in 10-fold series dilution, and 3 parallels were detected for each

concentration and repeated 3 times. The coefficient of variation intra-assay and inter-assay was calculated
to evaluate the reproducibility and stability of the method. 14 canine tissue samples and 66 porcine whole
blood samples were detected by the established quantitative PCR method and the PCR method
recommended by national standard to evaluate the capability of this method in practical application.
Result The established quantitative PCR method showed a linear relationship between Ct value and
plasmid concentration in the range of 108~ 101copies/ μL, the standard curve Slope was - 3. 285, R
2 value was 1, and the amplification efficiency was 101. 663%. The lowest detectable concentration of
Toxoplasma gondii was 101copies/ μL. There was no cross reaction with other 14 common swine
pathogens. Replicates were detected at 106 to 102 copies/ μL plasmid standards, and the coefficient of
variation intra-assay and inter-assay was less than 1. 21% and 0. 62%, respectively. The established
quantitative PCR method and the national standard PCR method were used to detect the positive rate of
14 canine tissue samples and 66 porcine whole blood samples. The result showed that the positive rate of
the established quantitative PCR method and the national standard PCR method were 10% ( 8 / 80) and
7. 5% ( 6 / 80 ) , respectively, and the positive coincidence rate reached 100%. Conclusion The
established Toxoplasma gondii FQ-PCR method can effectively detect Toxoplasma gondii. It provides a
good method for daily monitoring of toxoplasma gondii in laboratory animals.

Key words: Toxoplasma gondii, FQ-PCR, application

中图分类号:

Q95-3

罗霆宇李凯丽李昌文陈洪岩夏长友高彩霞.

实验动物弓形虫荧光定量 PCR 检测方法的建立及应用

[J]. 实验动物科学, 2024, 41(4): 8-14.

LUO Tingyu, LI Kaili, LI Changwen, CHEN Hongyan, XIA Changyou, GAO Caixia.

Establishment and Application of Fluorescent Quantitative PCR Assay for

Laboratory Animal Toxoplasma gondii

[J]. Laboratory Animal Science, 2024, 41(4): 8-14.

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实验动物弓形虫荧光定量 PCR 检测方法的建立及应用

Establishment and Application of Fluorescent Quantitative PCR Assay for

Laboratory Animal Toxoplasma gondii

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