Parasites
In the current work, the strain of T. vaginalis was isolated from the vaginal secretions of female patients clinically exhibiting certain trichomoniasis symptoms in the Third Affiliated Hospital of Xinxiang Medical University. The culture of T. vaginalis was performed using 10% calf serum, TYM medium added with antibiotics (100 mg/ml ceftriaxone, 50 mg/ml ciprofloxacin), fungicides (2.5 mg/ml amphotericin B) in a humidified chamber containing 5% CO2 at 37 ℃. The stationary phase parasites (2 × 106 parasites) were collected using a centrifuge and used subsequently in the experiments.
DNA extraction of T. vaginalis
PBS (pH 7.2) was used to wash the T. vaginalis trophozoite for thrice, which was subsequently isolated by centrifugation under the velocity of 5000 rpm for five minutes and subjected to DNA extraction using commercial kits (OMEGA) in accordance with the instruction.
The DNA simple of Trichinella spiralis, Toxoplasma gondii, Escherichia coli, Candida albicans, Staphylococcus aureus, Haemophilus were stored in Xinxiang Key Laboratory of Pathogenic Biology.
Detection of actin gene from T. vaginalis using nest-PCR
DNA extracts were subjected to species-specific nested PCR for T. vaginalis actin (GenBank Accession No. AF237734). Briefly, the primary PCR was performed in a 25 μL reaction mixture consisting of 12.5 μL of 2×PCR Master Mix (YIFEI XUE BIOTECHNOLOGY, Nanjing, China), 1 μL (0.1 μmol/L) each of the primers OUT-F and OUT-R (Table 1), 1.0 μL of DNA template (100-200ng), and 9.5 μL of double distilled water. The procedures and conditions of amplifying DNA included: three minutes at 95 ℃ for denaturation, 95 ℃ for 45 s, 55 ℃ for 30 s, and 72 °C for 1 min through 35 cycles, and 72 ℃ for 10 minutes for final extension. Then the second PCR was performed as the primary PCR, and the products of primary PCR and IN-F and IN-R (Table 1) were used as template and primers in this reaction. Electrophoresis using 1.5% agarose stained by 1.0 μL of Goldview Safe DNA Gel Stain (Yifei Xue Biotechnology, Nanjing, China) in advance was used to resolve the obtained DNA sequences at 100 V and a UV transilluminator (UVP, Upland, CA, USA) was used to visualize the bands.
Designing the primers of LAMP
LAMP Designer version 1.02 (Premier Biosoft, Palo Alto, CA, USA) was used to design the specific LAMP primers of T. vaginalisAP65 on the basis of the sequence in NCBI (GenBank Accession No. U35243.1). Four primers including outer primers (AP65-F3, AP65-B3), inner and forward primers (AP65-BIP, AP65-FIP) were chosen (Table 1). BLAST search was used to assess the specificity of these primers towards the sequences of target genes recorded by NCBI (http://www.blast.ncbi.nlm.nih.gov).
Detection of AP65 gene of T. vaginalis by LAMP assays
The mixed reaction solution with the volume of 20 μL which contained Betaine (5M) 6 μL (Sigma, St. Louis, MO, USA), 10×Bst DNA polymerase Buffer 2 μL, dNTPs Mixture (10mM each) 2 μL, each of AP65-FIP and AP65-BIP (16μM) 1 μL, each of F3 and B3 (4μM) 1 μL, MgCl2 (25mM) 3 μL, 8.0 U Bst DNA polymerase (TaKaRa, Dalian, China) 1.5 μL, and ddH2O 1.5 μL was used to perform the LAMP assays. The temperature condition of the LAMP reaction system was 63 ℃ for 120 minutes during the incubation and 80 ℃ for 10 minutes during the termination in a heating block. The experiment contained negative control (ddH2O) and positive control (T. vaginalis DNA).
The LAMP products were assessed by gel electrophoresis and addition of SYBR GreenⅠ(Solarbio, Beijing, China). 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 101 -1012 times and kept for use. The trophozoites of T. vaginalis were diluted to 103, 102, 101 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 (ddH2O) and positive control (T. vaginalis DNA). All the experiments were conducted in triplicate independently.