Development of a convenient detection method for Trichomonas vaginalis based on loop-mediated isothermal amplification targeting adhesion protein 65

Abstract

Background: Trichomoniasis resulting from Trichomonas vaginalis (T. vaginalis) has been considered as a commonly seen disease with the transmission way of sex. At present, the detection methods of T. vaginalis mainly include wet mount microscopy, culture, PCR, immunofluorescence and ELISA. However, all of these detection methods exist shortcomings. Methods: In this study, a loop-mediated isothermal amplification (LAMP) assay that targeted the species-specific sequence of adhesion protein 65 (AP65) gene had been conducted to detect T. vaginalis. The optimum reaction system and conditions were optimized in this rapid detection method. Results: The results of sensitivity analysis showed that the LAMP assay targeting the AP65 gene was 1000 times more sensitive than the nested PCR targeting the actin gene commonly used for detection of T. vaginalis, and the detecting limitation of the former was 10 trichomonad. Moreover, the amplification of the target gene AP65 by LAMP assay exhibited high specificity and the product was exclusively from T. vaginalis. The detection technique of LAMP did not exhibit cross-reactivity with the common pathogens of Trichinella spiralis, Toxoplasma gondii, Escherichia coli, Candida albicans, Staphylococcus aureus, Haemophilus. Conclusions: According to the present study, the LAMP assay with the target of AP65 gene, was suitable for the early diagnosis of T. vaginalis infections. Consequently, the LAMP assay was proposed by the current study as a point-of-care examination and an alternative molecular tool which exhibited the potential value in the treatment, control and prevention of trichomoniasis transmission and relevant complication.

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

“…However, in this research, we developed the RPA assay for diagnosis of T. vaginalis infection that only took 30 min to amplify the detectable amount of DNA. Moreover, the RPA assay successfully amplified T. vaginalis DNA in the temperature range of 33 to 45°C, which indicated that we can amplify the target gene by RPA using a simple heating device, such as a batterypowered heater, chemical heater, or even body heat (Lillis et al, 2014).…”

“…These samples were examined by wet mount microscopy, culture, nested PCR, and RPA, respectively. The cultivation process of T. vaginalis was described as follows: the clinical samples (50 μl) were inoculated into fresh TYM medium and cultured in a 37°C incubator containing 5% CO 2 for 72 h. As described in our previous article (Li et al, 2020), the actin gene of T. vaginalis was amplified by nested PCR from genomic DNA. The positive rate of T. vaginalis detected by these methods was calculated.…”

“…The quantity of infected people all around the world approximately reaches 270 million each year (Aquino et al, 2020). In the United States, roughly five million people were diagnosed with T. vaginalis infection annually, and the infection rate was 24.3% in Japan (Li et al, 2020). Moreover, the South Africa has approximately 30 million populaces with getting T. vaginalis infection every year (Mabaso & Abbai, 2021).…”

“…In China, the prevalence fluctuated greatly in disparate places (Liu et al, 2017;Lu et al, 2022;Luo et al, 2016;Zhang et al, 2018). T. vaginalis infection can cause a variety of inflammatory infections, such as trichomonas vaginitis, cervicitis, atypical pelvic inflammation (Patel et al, 2018), urethritis, and prostatitis (Van Gerwen et al, 2021), and can also lead to premature rupture of membranes, preterm birth, abortion, and low birth weight in pregnant women (Op de Coul et al, 2021;Zhang et al, 2020). Meanwhile, the extensive infection of T. vaginalis is related to the increased risk of human immunodeficiency virus infection Muzny et al, 2016) and cervical cancer (Yang et al, 2018).…”

“…At present, the main diagnostic methods of T. vaginalis are wet mount microscopy, culture, nested PCR, and loop‐mediated isothermal amplification (LAMP; Asmah et al, 2018; Hobbs & Seña, 2013; Li et al, 2020). Among these diagnostic methods, the wet mount microscopy is a common method for clinical diagnosis of female trichomoniasis.…”