Determination of Trichomonas vaginalis Genotypes Using PCR-Restriction Fragment Length Polymorphism (RFLP)

Demirag, Serpil; Malatyalı, Erdoğan; Ertuğ, Sema; Ertabaklar, Hatice

doi:10.5152/tpd.2017.5496

Cited by 9 publications

(5 citation statements)

References 18 publications

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“…Lee et al established a PCR method using E650 as a target gene for diagnosis of T. vaginalis infection among male patients with chronic recurrent prostatitis and urethritis (Lee et al, 2012). Demirağ et al developed a PCR method targeting the actin gene of T. vaginalis , which was used to diagnose early infections of T. vaginalis (Demirağ et al, 2017). In this study, we found that amplification of actin gene with the primer pairs of Actin‐F/‐R was the most efficient for detection of T. vaginalis by RPA.…”

Section: Discussionmentioning

confidence: 99%

Construction a novel detection method for Trichomonas vaginalis based on recombinant enzyme polymerase amplification targeting the Actin gene

Deng

Sheng

et al. 2023

J Eukaryotic Microbiology

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Trichomoniasis is a common and curable sexually transmitted disease worldwide. The rapid, convenient, and accurate diagnosis of trichomoniasis is an important link in the prevention and treatment of the disease. The current detection methods of Trichomonas vaginalis are mainly wet mount microscopy, culture, nested PCR, and loop‐mediated isothermal amplification. However, these detection methods have some shortcomings. In this study, a recombinant enzyme polymerase amplification (RPA) assay had been conducted to detect T. vaginalis. The target gene and the corresponding primers were screened, and the reaction system and conditions were optimized in the assay of RPA. The sensitivity and specificity of this detection method were analyzed. The detection efficiency of wet mount microscopy, culture, nested PCR, and RPA was compared by testing 53 clinical samples from vaginal secretions. By screening, the actin gene of T. vaginalis could be used as a target gene for RPA detection of T. vaginalis, and the optimum reaction condition to amplify the actin gene by RPA was at 39°C for 30 min. The detection limit of T. vaginalis DNA using RPA was 1 pg, corresponding to a sensitivity of approximately five trophozoites. The RPA assay demonstrated high specificity for T. vaginalis, and there was no cross‐reactivity with Giardia lamblia, Escherichia coli, Lactobacillus, Toxoplasma gondii, Staphylococcus aureus, and Candida albicans. Of the 53 clinical samples, the positive rates of T. vaginalis detected by wet mount microscopy, culture, nested PCR and RPA were 50.9 4% (27/53), 71.7% (38/53), 71.7% (38/53), and 69.81% (37/53), respectively. Compared with culture which was used as the gold standard for diagnosing trichomoniasis, testing clinical samples by wet mount microscopy showed 71.05% sensitivity, 100% specificity, and moderate diagnostic agreement with the culture (K = 0.581, Z = 4.661, p < 0.001). The nested PCR showed 100% sensitivity, 100% specificity, and excellent diagnostic agreement (K = 1, Z = 7.28, p < 0.001), while RPA displayed 97.37% sensitivity, 100% specificity, and excellent diagnostic agreement (K = 0.954, Z = 6.956, p < 0.001). At the present study, rapid amplification of actin gene by RPA could be used as a tool for detection of T. vaginalis. The detection method of RPA was more sensitive than wet mount microscopy and displayed excellent specificity. Moreover, RPA amplification of actin gene did not require a PCR instrument and the amplification time was shorter than that of ordinary PCR. Therefore, the RPA assay was proposed in this study as a point‐of‐care examination and a diagnostic method of T. vaginalis infection, which exhibited the potential value in the treatment and prevention of trichomoniasis.

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Section: Discussionmentioning

confidence: 99%

Construction a novel detection method for Trichomonas vaginalis based on recombinant enzyme polymerase amplification targeting the Actin gene

Deng

Sheng

et al. 2023

J Eukaryotic Microbiology

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“…PCR-RFLP based on the actin gene has been extensively studied for the genotyping of T. vaginalis strains. In Turkey, the majority of T. vaginalis isolates were the actin genotype E (45%), the remaining isolates were genotype G (7.5%), genotype N (1.5%), and genotype H (1.5%), two of which were mixed genotypes E and H (10%) [ 40 ]. In Kenya, five actin genotypes were revealed by RFLP analysis, and the researchers used the latter method to detect five genotypes: E (50%), G (13.6%), I (4.5%), N (27.3%), and P (4.5%) [ 41 ].…”

Section: Discussionmentioning

confidence: 99%

Prevalence and Genotype of Trichomonas vaginalis among Men in Xinxiang City, Henan Province, China

Zhang

Sang

et al. 2023

Journal of Tropical Medicine

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Trichomonas vaginalis (T. vaginalis) could cause trichomoniasis through sexual transmission, which was globally distributed. In this study, the prevalence and phylogenetic analyses of T. vaginalis among men in Xinxiang were conducted. From October 2018 to December 2019, a total of 634 male clinical samples were collected, including 254 samples of semen, 43 samples of prostate fluid, and 337 samples of urine. These samples were examined by nested PCR and a total of 32 (5.05%) T. vaginalis-positive samples were detected. Among these samples, the positive rates of T. vaginalis in semen, prostate fluid, and urine were 7.87% (20/254), 4.65% (2/43), and 2.97% (10/337), respectively. Three actin genes were successfully isolated and sequenced from the 32 positive DNA samples, and the analysis of the sequence and phylogenetic tree showed that the three actin gene sequences exhibited 99.7%–100% homology to the published actin gene sequence (EU076580) in NCBI, and the T. vaginalis strains in the three positive samples were identified as genotype E. Our results demonstrate a notable genotype of T. vaginalis in the male population and provide insight into the performance of these genetic markers in the molecular epidemiology of trichomoniasis. However, further studies are needed to research the association between the genotype and the pathogenicity of T. vaginalis.

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“…Except using wet mount microscopy, staining techniques, several methods also have used for detecting trichomoniasis, including culture, immunodiagnosis, and molecular diagnosis. [7,8,9,10].Kinds of genes or tools have used to study teh genetic properties of T.vaginalis, including actin gene, RFLP, ITS, qRT-PCR, PCR sequencing and so on [11,12,13,14] . Several researchers using microsatellites and single-copy genes acted polymorphic makers to study genetic diversity in T.vaginalis, and has been mainly identified two population [15].…”

Section: Introductionmentioning

confidence: 99%

comparison of genetic diversity and phylogenetic structure of 18S gene ofTrichomonas vaginalisin Hainan and Xinjiang provinces

Liu,

Dong,

Wang

et al. 2024

Preprint

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Trichomonas vaginalisis a kind of flagellate parasite endemic all of the world. study compared the difference of genetic diversity and phylogenetic structure of 18S ribosomal RNA gene ofT. vaginalisfrom Hainan and Xinjiang Provinces, China. 20 samples and 47 samples from Hainan and Xinjiang respectively which confirmed for infection with T.vaginalis. The sequences were aligned using MEGA 11 software and phylogenetic trees were drawn by Neighbor-Joining method. The number of mutation, nucleotide diversity, and haploid diversity were analyzed using Dnaspv5 software. For Hainan samples, the analyses showed 31 polymorphisms, creat different haplotypes with a haplotype diversity of 0.589. Nucleotide diversity and average nucleotide different amongT.vaginaliswere estimated as 0.00679 and 3.447, respectively.Tajima’s D value in Tajiama’s neutrality test was -2.48147,which was significant (p<0.001). But for Xinjiang samples, the analyses showed 31 polymorphisms, creat different haplotypes with a haplotype diversity of 0.731. Nucleotide diversity and average nucleotide different among T.vaginalis were estimated as 0.00497 and 2.50435, respectively. Tajima’s D value in Tajiama’s neutrality test was -2.26284, which was significant (p<0.01). For all the sequence in this study, all the sequences form one phylogenetic tree, some of the sequence from two provinces showed a certain aggregating phenomenon. The differences between the samples from two province maybe for the local environment, ethnic, sample number and so on, it need further study for T.vaginalis.

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Determination of Trichomonas vaginalis Genotypes Using PCR-Restriction Fragment Length Polymorphism (RFLP)

Cited by 9 publications

References 18 publications

Construction a novel detection method for Trichomonas vaginalis based on recombinant enzyme polymerase amplification targeting the Actin gene

Construction a novel detection method for Trichomonas vaginalis based on recombinant enzyme polymerase amplification targeting the Actin gene

Prevalence and Genotype of Trichomonas vaginalis among Men in Xinxiang City, Henan Province, China

comparison of genetic diversity and phylogenetic structure of 18S gene ofTrichomonas vaginalisin Hainan and Xinjiang provinces

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