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

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.

. For gel electrophoresis, 1.5% agarose gel was used to resolve 5.0 μL of the product of LAMP, as mentioned in Section 2.4, and the positive result was indicated by the pattern of bands like ladders. For addition of SYBR GreenⅠ, 2.0 μL of 1000× SYBR GreenⅠwas added to the remainder 15 μL of LAMP products, and the positive result was revealed by dull to fluorescent signal or change of colors from orange to green under visible light.

Analytical sensitivity of detection of AP65 gene of T. vaginalis by LAMP assay
The DNA with the initial concentration of 90 ng/μL in 2.3 was diluted by 10 1 -10 12 times and kept for use. The trophozoites of T. vaginalis were diluted to 10 3 , 10 2 , 10 1 and 1 respectively, and the DNA of these diluted samples was extracted. Then, the prepared DNA samples were amplified by nested PCR and LAMP respectively, as described above. All the experiments were conducted in triplicate independently.

Analytical specificity of detection of AP65 gene of T. vaginalis by LAMP assay
The examination of the analytical specificity of AP65 gene of T. vaginalis by LAMP assay was conducted for determining the cross-reactive degree among DNA extraction samples from various trichomonads, Trichinella spiralis, Toxoplasma gondii, Escherichia coli, Candida albicans, Staphylococcus aureus and Haemophilus with close relation. The experiment contained negative control (ddH 2 O) and positive control (T. vaginalis DNA). All the experiments were conducted in triplicate independently.

Detecting T. vaginalis by nested PCR and LAMP
T. vaginalis actin gene was amplified using nest PCR with specific primers, and a positive sequence of 1100 bp was obtained in line with the expectation (Fig.1A). There were no target amplicons in negative controls.
According to the results of electrophoresis, the products amplified by LAMP with the target of AP65 in T. vaginalis exhibited distinctive pattern of multiple bands (Fig.1B). An orangeto-green color change was observed under natural light or a dull to fluorescent signal under UV transilluminator, after addition of SYBR GreenⅠ (Fig.1C).

The analytical sensitivity of T. vaginalis nested PCR and LAMP
The initial concentration of 90 ng/μL of T. vaginalis DNA was diluted by 10 1 -10 12 times. As shown in Fig.2, the lowest template concentration was 90 × 10 -7 ng/μL detected by nested PCR ( Fig.2A and 2B), while that was 90 × 10 -10 detected by LAMP amplification (Fig.2C and   2D). It could be seen that the analytical sensitivity of LAMP assays of T. vaginalis established in this study was 1000 times higher than that of nested PCR.
In order to further explore the sensitivity of LAMP amplification, the trophozoites of T.
vaginalis were diluted to 10 3 , 10 2 , 10 1 and 1 respectively, and the DNA of these diluted samples was extracted. These results were shown that 10 3 trophozoites of T. vaginalis could be detected by nested PCR (Fig.3A and 3B), while 10 trophozoites of T. vaginalis could be detected by LAMP amplification (Fig.3C and 3D).

The analytical specificity of LAMP for T. vaginalis
There were no cross reactions in the LAMP using DNA extractions from T. vaginalis and other microorganisms including Trichinella spiralis, Toxoplasma gondii, Escherichia coli, Candida albicans, Staphylococcus aureus and Haemophilus. There were objective bands exclusively in experiments using DNA from T. vaginalis, either the results were observed under visible light by adding SYBR GreenⅠor using a UV transilluminator (Fig. 4).

Discussion
As a kind of protozoan parasite that infects the genito-urinary system of human beings, T.
vaginalis is found worldwide but receives less attention compared to other agents of STIs compared to conventional PCR [40][41][42]. A LAMP assay designed to detect Cyrptosporidium parvum was even proven to have a 100000 fold lower limit of detection compared to PCR [43]. The LAMP assay exhibited 1000 times higher sensitivity than the nested PCR in amplifying target genes of T. vaginalis. According to the results, the detecting limitation of LAMP was 10 trophozoites of T. vaginalis and that of nested PCR was as many as 10 3 , and the difference was possibly due to the Bst DNA polymerase which was highly tolerate to inhibitors of nucleic acid amplification tests. Toye et al indicated that PCR was rarely inhibited using urine specimens, infrequently inhibited using endocervical swabs while frequently inhibited using urethral swabs [44]. Although the inhibiting effect could be reduced by various preparing approaches, it is not sufficient to eliminate the inhibitors of PCR from genital swabs by boiling the specimens. Moreover, for LAMP there was no decrease of signal intensity up to 100 trichomonads/mL and the bands in gel with stains of SYBR Safe exhibited typical ladder-like pattern, possibly resulting from significantly higher amplifying product quantity in comparison to that of PCR [38]. As complicated body fluid, urine and genital secretion had been applied to detecting T. vaginalis on the basis of nucleic acid, and the sensitivity of which were 64% -100% for urine samples and 81% -100% to genital secretion swabs [45,46]. Previous studies have shown that LAMP reactions were not inhibited in spiked urine specimens [47]. Nevertheless, due to the usage of the wild type enzyme in the LAMP approach, unwanted activity of DNA polymerase in the process of setup possibly led to fail in reproducible amplification. In order to solve this problem, a warm start strand-displacing DNA polymerase which kept stable under ambient temperature while didn't work until 50 °C was used by Tanner et al [48]. In our study, the time cost for performing LAMP of T. vaginalis with high analytical sensitivity from preparing samples to detecting LAMP products was no longer than 130 minutes.
Although some studies showed that LAMP could detect trichomonad based on target genes of actin, 18S rRNA and the 2-kbp repeated DNA, these genes have relatively high homology among different species [49][50][51]. AP65 is a prominent adhesin of T. vaginalis that mediates binding of parasites to host vaginal epithelial cells (VECs) [52]. We found that the sequences of AP65 had almost no homology with other species. Accordingly, AP65 as the target gene for detection possessed excellent specificity. In this current study, the LAMP of T. vaginalis was highly specific without crossreactivity to trichomonads of human beings with close relationship, such as Trichinella spiralis, Toxoplasma gondii, Escherichia coli, Candida albicans, Staphylococcus aureus and Haemophilus. Many previous studies shown that 6 primers were used for LAMP amplification of target genes [53,54]. However, in the research, the 4 primers that recognized 6 instinct sites of the target gene led to the high specificity of LAMP, and AP65 gene could be detected specifically and sensitively without primers LF and LB. Therefore, the AP65 gene of T. vaginalis which was selected as the target gene for conducting highly specific LAMP could be used in the diagnosis of trichomoniasis [55,56].
Nevertheless, there are a few shortages of LAMP. In spite of the high sensitivity and specificity, it is possible for the large quantity of DNA sequences obtained through the experiment to be contaminated by the opened tubes, which results in fault-positive consequences. In most cases, staining materials of nucleic acids , such as SYBR GreenⅠand Pico Green, are used in LAMP for detecting the product, while these materials exert an inhibiting effect on the amplifying process of LAMP. In addition, more than 1% contamination of biological substances such as blood and urine or more than 30% of saline solution or PBS can inhibit LAMP as well as PCR reactions [57]. Raw milk contaminants can even inhibit LAMP but not PCR. Consequently, using a real-time turbimeter to monitor the reaction in close tubes [38], using hydroxylnaphthol blue dye added in advance to conduct  Table   Table 1 Oligonucleotide primer sequences used for Nested PCR in this research.   The template DNA for LAPM amplification was from T. vaginalis, Trichinella spiralis, Toxoplasma gondii, Escherichia coli, Candida albicans, Staphylococcus aureus and Haemophilus respectively.