CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >
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)
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
LIU Ji-bing , ZHAO Hong-xi . Development of PCR diagnostic method for Trichomonas foetus infection based on 18S rRNA[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022 , 40(5) : 682 -685 . DOI: 10.12140/j.issn.1000-7423.2022.05.019
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