收稿日期: 2022-02-25
修回日期: 2022-07-28
网络出版日期: 2022-10-25
基金资助
宁夏自然科学基金(2021AAC03011)
Development of PCR diagnostic method for Trichomonas foetus infection based on 18S rRNA
Received date: 2022-02-25
Revised date: 2022-07-28
Online published: 2022-10-25
Supported by
Ningxia Natural Science Foundation(2021AAC03011)
根据GenBank已发表的胎毛滴虫18S rRNA基因序列设计合成引物,建立胎毛滴虫PCR检测方法。以胎毛滴虫阳性重组质粒pUCm-T-TF18S为模板,以猫贾第虫基因组DNA、猫球虫基因组DNA、猫细小病毒基因组DNA、猫冠状病毒基因组cDNA为对照进行PCR检测,分析该方法的特异性。将胎毛滴虫阳性重组质粒以10倍为梯度稀释成8个不同浓度,进行PCR检测,分析该方法的敏感性。取分别置于-20 ℃ 3、6、9、12个月的pUCm-T-TF18S质粒进行PCR检测,分析该方法的稳定性。用建立的PCR检测方法对20份猫粪样进行检测,并与镜检结果进行对比。结果显示,重组质粒pUCm-T-TF18S PCR扩增后出现特异性条带。测序结果表明,产物序列长度为1 264 bp,经BLAST序列比对分析,与猫源胎毛滴虫（GenBank登录号M81842.1）序列一致性为99.53%。建立的胎毛滴虫PCR检测方法与猫球虫、猫贾第虫、猫冠状病毒、猫细小病毒均无交叉反应;可检测的最低模板浓度为4.52 × 105 拷贝/μl;胎毛滴虫pUCm-T-TF18S质粒在-20 ℃冰箱放置12个月后仍可检测出目的条带。该方法从20份猫粪样中检出16份胎毛滴虫核酸阳性,较传统显微镜检查结果（13份）更为准确、敏感。提示建立的胎毛滴虫PCR检测方法特异性强、敏感性高、稳定性强,可用于临床上胎毛滴虫的检测与流行病学的调查。
刘继兵 , 赵洪喜 . 基于18S rRNA的胎毛滴虫感染PCR诊断方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2022 , 40(5) : 682 -685 . DOI: 10.12140/j.issn.1000-7423.2022.05.019
To establish a PCR detection method for Trichomonas foetus, the primers were designed and synthesized according to the 18S rRNA gene sequence of T. foetus published by GenBank. The positive recombinant plasmid pUCm-T-TF18S of T. foetus was used as the template, and the genomic DNA of Giardia felis, Coccidia felis, feline parvovirus and cDNA of feline coronavirus were used as the control for PCR detection to analyze the specificity of this method. The positive T. foetus recombinant plasmid was serial to 8 different concentrations with a gap of 10 folds, and PCR was performed to analyze the sensitivity of this method. The pUCm-T-TF18S plasmids stored at -20 ℃ for 3, 6, 9 and 12 months were detected by PCR to analyze the stability of the method. Twenty cat fecal samples were tested using this established PCR assay and compared with those of microscopic examination. The results showed that the recombinant plasmid pUCm-T-TF18S gave specific bands after PCR amplification. The sequencing results showed that the length of the product sequence was 1 264 bp, and the BLAST sequence comparison analysis showed 99.53% sequence identity, which is consistent with that of T. foetus from cats (GenBank registration number M81842.1). The PCR method for detection of T. foetus had no cross-reactivities with C. felis, G. felis, feline coronavirus and feline parvovirus; the minimum detectable template concentration is 4.52 × 105 copies/μl; The target band of T. foetus DNA can still be detected after being stored in the refrigerator at -20 ℃ for 12 months. This method detected 16 positive samples of T. foetus nucleic acid from 20 cat fecal samples, which is more accurate and sensitive than the results from traditional microscopy (13 samples). It is suggested that the PCR method for the detection of T. foetus is highly specific, sensitive and stable, and can be used for clinical detection and epidemiological investigation of T. foetus.
Key words： Cat; Trichomonas foetus; PCR; Diagnostic method
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