PCR method for simultaneously detecting pentatrichomonas hominis and tetratrichomonas buttreyi

Abstract

The present invention relates to the technical field of molecular detection. Provided is a duplex nested PCR method capable of simultaneously detecting Pentatrichomonas hominis and Tetratrichomonas buttreyi. A positive control mixture of Pentatrichomonas hominis and Tetratrichomonas buttreyi is used as a template, and a Trichomonas genus-specific primer is used as an amplification primer to perform amplification; and an amplification product obtained from the first round of PCR is used as a template, and a mixture of a Pentatrichomonas hominis-specific primer and a Tetratrichomonas buttreyi-specific primer is used as a primer to perform amplification, thereby enabling simultaneous detection of Pentatrichomonas hominis and Tetratrichomonas buttreyi. The method can specifically amplify target fragments of Pentatrichomonas hominis and Tetratrichomonas buttreyi, and can detect positive control DNA at a minimum concentration of 500 fg / μL.

Description

PCR method for simultaneously detecting human pentatrichomonas and battrey tetratrichomonas TECHNICAL FIELD

[0001] The present application belongs to the field of molecular detection technology, and relates to a duplex PCR method for simultaneously detecting human pentatrichomonas and battrey tetratrichomonas and application thereof. BACKGROUND

[0002] Trichomonas is a protozoan that parasitizes the digestive system and urogenital tract of vertebrates, is the oldest eukaryote, and includes both pathogenic species and some symbiotic species. Trichomonas is also a very common protozoan in animals, and currently reported trichomonas species mainly include Trichomonas vaginalis (T. vaginalis), Tritrichomonas foetus (T. foetus), Trichomonas gallinae (T. gallinae), Pentatrichomonas hominis (P. hominis), and Tetratrichomonas buttreyi (T. buttreyi). Among them, T. foetus is the most common bovine trichomonas, parasitizes the reproductive organs of cows and bulls, and can cause a kind of sexually transmitted protozoan disease in cattle, i.e., bovine trichomoniasis, which can cause premature birth and stillbirth in cattle, and further cause significant economic losses to the cattle industry. The disease has been included in the OIE list of World Organization for Animal Health diseases, and has been listed as a category III animal disease in China. P. hominis can breed in the large intestines of humans, cattle, pigs, deer, raccoon dogs, dogs, cats, non-human primates and rodents, is excreted out of the body through feces, and trophozoites can be directly transmitted between hosts. It was generally believed in the past that P. hominis and the host were in a symbiotic relationship, but some reports believe that P. hominis is one of the causes of diarrhea in human or animal hosts, and the potential risk of human-animal co-infection still needs to be further determined. T. buttreyi is a symbiotic trichomonas that widely parasitizes the intestines of pigs, cattle, raccoon dogs, deer and other animals, and the infection rate is higher in young and old livestock groups.

[0003] The infection of animals with trichomonad is mostly latent, even if there are clinical symptoms, the symptoms are often not typical, so the diagnosis of trichomoniasis is difficult, and the final diagnosis must rely on laboratory detection. The current routine methods for detecting trichomonad mainly include light microscopy, culture and molecular detection. However, the morphologies of different trichomonad species are similar, and the different trichomonad species cannot be accurately distinguished under a light microscope after light microscopy or culture. In cattle, due to defecation or occasional physical contact between the cow and the bull, intestinal trichomonads such as human five trichomonads and Barth trichomonads may contaminate the reproductive tract of the cow and survive in the reproductive tract for a short period of time, which may cause false positive results when detected by microscopy or culture, and further overestimate the prevalence of fetal three trichomonads in the cattle population. In addition, light microscopy and culture require that the samples such as feces or reproductive tract flushes must be fresh, and there are disadvantages such as false negatives caused by sample death due to refrigeration or lack of timely processing of samples. At present, some molecular methods for detecting fetal three trichomonads have been developed, but there are few reports on molecular methods for detecting human five trichomonads and Barth trichomonads.

[0004] SUMMARY

[0005] The technical problem to be solved by the present application is to provide a duplex nested PCR method for simultaneously detecting human five trichomonads and Barth trichomonads in the feces of multiple hosts, which specifically amplifies the target fragments of human five trichomonads and Barth trichomonads by two rounds of nested PCR, and has no cross reaction with the genomic DNA of multiple protozoa such as fetal three trichomonads, microcryptosporidium, giardia, blastocystis, intestinal microsporidium, bovine eimeria and que's eimeria, and can detect 500 fg / μL of positive control DNA at the minimum.

[0006] The present application provides a duplex nested PCR method for simultaneously detecting human five trichomonads and Barth trichomonads, which specifically comprises the following steps:

[0007] S1, first round PCR: using human five trichomonads and Barth trichomonads positive control mixture as template, and using trichomonas-specific primers as amplification primers for amplification;

[0008] S2, second round PCR: using the amplification product obtained in S1 as template, and using a mixture of human five trichomonad-specific primers and Barth trichomonad-specific primers as primers for amplification;

[0009] Amplification obtains two target fragments with sizes of 339 bp and 623 bp, i.e. human five trichomonads and Barth trichomonads are simultaneously detected.

[0010] Further, in S1, the sequence of the upstream primer F of the Trichomonas-specific primer is shown in SEQ ID No. 1, and the sequence of the downstream primer R is shown in SEQ ID No. 2.

[0011] Further, in S2, the Trichomonas parabursari-specific primer comprises an upstream primer TF with the sequence shown in SEQ ID No. 3 and a downstream primer TR with the sequence shown in SEQ ID No. 4.

[0012] Further, in S2, the Trichomonas hominis-specific primer comprises an upstream primer PF with the sequence shown in SEQ ID No. 5 and a downstream primer PR with the sequence shown in SEQ ID No. 6.

[0013] Further, in S1, the PCR reaction condition is 95℃ for 10 min, then 95℃ for 60 s, 60℃ for 60 s, 72℃ for 60 s, for a total of 30 cycles, and finally 72℃ for 7 min.

[0014] Further, in S2, the PCR reaction condition is 95℃ for 10 min, then 95℃ for 60 s, 58℃ for 60 s, 72℃ for 60 s, for a total of 30 cycles, and finally 72℃ for 7 min.

[0015] Further, in S2, the 339bp-sized target fragment is Trichomonas hominis DNA.

[0016] Further, in S2, the 623bp-sized target fragment is Trichomonas parabursari DNA.

[0017] The application further discloses an application of the double nested PCR method for simultaneously detecting Trichomonas hominis and Trichomonas parabursari in the breeding of animals such as cattle, pigs, raccoon dogs, and deer.

[0018] Further, the double nested PCR method is used for simultaneously detecting Trichomonas hominis and Trichomonas parabursari in the feces of various animal hosts such as cattle, pigs, raccoon dogs, and deer.

[0019] Compared with the prior art, the application has the following beneficial effects:

[0020] 1. The double nested PCR method can specifically amplify the target fragments of Trichomonas hominis and Trichomonas parabursari, and has no cross reaction with the genomic DNA of various protozoa such as Trichomonas foetus, Cryptosporidium parvum, Giardia lamblia, Blastocystis hominis, Enterocytozoon bieneusi, Eimeria bovis, and Eimeria quail; and the method can detect 500 fg / μL of positive control DNA at the minimum.

[0021] 2. The duplex nested PCR method provided by the application can quickly, accurately and simply detect Trichomonas hominis and Trichomonas batrachii in feces of various animal hosts such as cows, pigs, raccoon dogs and deer, and can be directly used in clinical detection, and is of great significance for rapid detection and identification of the two kinds of trichomonads, epidemiological investigation of the trichomonads, and prevention and control of the trichomonads in breeding farms of various animals such as cows, pigs, raccoon dogs and deer.

[0022] 3. The duplex nested PCR method provided by the application has high specificity, and 1:10 5 The positive control DNA is diluted, and the detection sensitivity is as low as 500 fg / μL; the detection results of the duplex nested PCR method and the single nested PCR method are consistent, and there is no significant difference (P>0.05); and the duplex nested PCR method can significantly save test time and reduce test cost. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0024] Fig. 1 is a comparison of the amplification results of the single nested PCR and the duplex nested PCR in the embodiment of the application;

[0025] In the figure, the meanings of the lanes are as follows: M: DL2000 DNA Marker; 1: Trichomonas hominis single nested PCR; 2: Trichomonas batrachii single nested PCR; 3: negative control; 4: duplex nested PCR;

[0026] Fig. 2 is the specificity test result of Trichomonas hominis in the embodiment of the application;

[0027] In the figure, the meanings of the lanes are as follows: M: DL2000 DNA Marker; 1: double-distilled water; 2: Trichomonas hominis; 3: Trichomonas foetus; 4: Cryptosporidium parvum; 5: Giardia lamblia; 6: Blastocystis hominis; 7: Enterocytozoon bieneusi; 8: Eimeria bovis; 9: Eimeria cuvii;

[0028] Fig. 3 is the specificity test result of Trichomonas batrachii in the embodiment of the application;

[0029] Wherein, each lane meaning as follows: M: DL2000 DNA Marker; 1: double distilled water; 2: Babesia microti; 3: Trichuris foetus; 4: Cryptosporidium parvum; 5: Giardia lamblia; 6: Blastocystis hominis; 7: Enterocytozoon bieneusi; 8: Eimeria bovis; 9: Eimeria quaili;

[0030] Figure 4 is the result of double nested PCR product amplification specificity test in the embodiment of the present application;

[0031] Wherein, each lane meaning as follows: M: DL2000 DNA Marker; 1: Trichuris foetus; 2: Cryptosporidium parvum; 3: Pentatrichomonas hominis; 4: Babesia microti; 5: Pentatrichomonas hominis + Babesia microti; 6: Giardia lamblia; 7: Blastocystis hominis; 8: Enterocytozoon bieneusi; 9: Eimeria bovis; 10: Eimeria quaili;

[0032] Figure 5 is the result of double nested PCR sensitivity test in the embodiment of the present application;

[0033] Wherein, each lane meaning as follows: M: DL2000 DNA Marker; 1: 1-8 are positive templates, 1-8 respectively represent 1:10 0 -1:10 7 times dilution, 9: blank control. DETAILED DESCRIPTION

[0034] The present application will be described in detail below with reference to the accompanying drawings and specific embodiments, but should not be understood as limiting the present application. If not specifically stated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0035] Example 1

[0036] I. Materials and methods

[0037] 1. Materials

[0038] (1) Worm DNA: The genomic DNA of Trichuris foetus, Cryptosporidium parvum, Giardia lamblia, Blastocystis hominis, Enterocytozoon bieneusi, Eimeria bovis, and Eimeria quaili was isolated and preserved by the laboratory. The positive controls of Pentatrichomonas hominis and Babesia microti were the recombinant plasmid DNA obtained by inserting the SSU rRNA fragments of bovine-derived Pentatrichomonas hominis and Babesia microti into pMD18-T vector, with a concentration of 50 ng / μL, prepared and preserved by the laboratory. The sequence of SSU rRNA of Pentatrichomonas hominis is shown in SEQ ID No. 7, and the sequence of SSU rRNA of Babesia microti is shown in SEQ ID No. 8.

[0039] (2) Collection of fecal samples for clinical detection: 300 fresh fecal samples were collected from multiple large-scale dairy farms (126 samples), yellow cattle farms (123 samples) and buffalo farms (51 samples), 500 fresh fecal samples were collected from multiple large-scale pig farms, 389 fresh fecal samples were collected from multiple captive raccoon breeding farms, and 336 fresh fecal samples were collected from multiple deer farms, each sample was about 100 g and was placed in a clean plastic bag, then was taken back to the laboratory and stored in a 4°C refrigerator for detection.

[0040] (3) Main reagents: rTaq DNA polymerase, PCR Buffer, dNTPs and DNA Marker DL2000 were products of Takara Company; fecal DNA extraction kit was a product of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0041] 2. Method

[0042] (1) Design and synthesis of primers

[0043] Table 1 PCR primers used in this example

[0044] The primers were designed according to the SSU rRNA genes of Pentastoma americana and Batraletta quadrioculata, as shown in Table 1, and were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0045] (2) PCR amplification

[0046] (2.1) Nested PCR of Pentastoma americana

[0047] The reaction system is shown in Table 2, the first round of PCR used Pentastoma americana positive control as template, and the primer was the Trichodinidae-specific primer in Table 1, the second round of PCR used the first round of PCR reaction product as template, and the primer was the Pentastoma americana species-specific primer in Table 1.

[0048] (2.2) Nested PCR of Batraletta quadrioculata

[0049] The reaction system is shown in Table 2, the first round of PCR used Batraletta quadrioculata positive control as template, and the primer was the Trichodinidae-specific primer in Table 1, the second round of PCR used the first round of PCR reaction product as template, and the primer was the Batraletta quadrioculata species-specific primer in Table 1.

[0050] (2.3) Double nested PCR

[0051] The reaction system is shown in Table 2, the first round of PCR template is a mixture of positive controls of P. hominis and P. batheri, the primer is the Trichomonas-specific primer shown in Table 1, the second round of PCR template is the first round of PCR reaction product, and the primer is a mixture of P. hominis species-specific primer and P. batheri species-specific primer shown in Table 1.

[0052] (2.4) PCR reaction conditions

[0053] For both single nested PCR and double nested PCR, the first round of PCR reaction conditions are 95℃ for 10 min, then 95℃ for 60 s, 60℃ for 60 s, 72℃ for 60 s, for a total of 30 cycles, and finally 72℃ for 7 min. The second round of PCR reaction conditions are the same as the first round of reaction conditions, with the annealing temperature adjusted to 58℃, and the rest of the parameters unchanged.

[0054] Table 2 Trichomonas SSU rRNA gene PCR reaction system

[0055] (2.5) Detection

[0056] After the reaction is completed, 2.0% EB-added agarose gel is prepared, 20 μL of PCR product and 2 μL of 10× electrophoresis loading buffer are mixed and loaded, and stable voltage electrophoresis is performed at 114 V. When the bromophenol blue is about 1 cm away from the other end of the gel, stop electrophoresis, place it under the gel imaging system, observe, take pictures, and record the results.

[0057] (3) PCR reaction specificity test

[0058] (3.1) Single nested PCR specificity test

[0059] The first round of PCR uses P. hominis and P. batheri as templates, with the genomic DNA of Trichomonas, Cryptosporidium parvum, Giardia lamblia, Blastocystis hominis, Enterocytozoon bieneusi, Eimeria bovis, and Eimeria quaili, and double-distilled water as controls, and the primer pair is the Trichomonas-specific primer. The second round of PCR uses the first round of PCR product as the template and uses two Trichomonas species-specific primers for PCR to detect the specificity of single nested PCR.

[0060] (3.2) Double nested PCR specificity test

[0061] In the first round of PCR, two positive control DNA mixtures, namely *Trichomonas vaginalis* and *Trichomonas barleyi*, were used as templates. Genomic DNA from *Trichomonas fetus*, *Cryptospora microsporum*, *Giardia lamblia*, *Bacillus spp.*, *Microsporidium bifidum*, *Eimeria auricularia*, and *Eimeria churnii*, along with double-distilled water, were used as controls. *Trichomonas* genus-specific primers were used. In the second round of PCR, the products from the first round of PCR were used as templates. The two species-specific primers were used alone or in combination to perform PCR to identify their specificity.

[0062] (4) Sensitivity test of double nested PCR reaction

[0063] Dilute to a starting concentration of 50 ng / μL, and then use a 10-fold dilution method, diluting sequentially at a ratio of 1:10. 0 1:10 1 1:10 2 1:10 3 1:10 4 1:10 5 1:10 6 1:10 7 Serial dilutions were performed, and the positive control DNAs of Trichomonas vaginalis and Trichomonas barley were diluted to eight concentration gradients. 1 μL of each dilution was taken as the first-round PCR template for double nested PCR amplification. The amplification products were subjected to 1% agarose gel electrophoresis to detect their sensitivity.

[0064] (5) Stability test

[0065] Each experiment was repeated three times to verify the stability of the experimental method.

[0066] (6) Testing of clinical samples

[0067] Fresh fecal samples collected from cattle, pigs, raccoon dogs, and deer were used to extract genomic DNA using a fecal DNA extraction kit. The DNA was then detected using single nested PCR for detecting Trichomonas vaginalis and Trichomonas barbali, as well as a double nested PCR method. The experimental results were compared.

[0068] II. Results and Analysis

[0069] 1. DNA extraction results

[0070] The extracted Trichomonas positive control DNA was measured by spectrophotometer, and the OD260 / OD280 ratio was in the range of 1.8 to 2.0, which met the experimental requirements.

[0071] 2. Results of single-nested PCR and double-nested PCR amplification

[0072] As shown in Fig. 1, the single nested PCR of Pentatrichomonas hominis and the single nested PCR of Battrachomonas hominis can specifically amplify the target fragments of Pentatrichomonas hominis and Battrachomonas hominis respectively; meanwhile, the double nested PCR can specifically amplify the target fragments of Pentatrichomonas hominis and Battrachomonas hominis simultaneously.

[0073] 3. The results of PCR specificity test

[0074] (1) The results of single PCR specificity test

[0075] As shown in Fig. 2, using the single nested PCR method of Pentatrichomonas hominis, after two rounds of PCR amplification, only the bovine-derived Pentatrichomonas hominis positive control as template can amplify a specific target band with the expected size of 339 bp, and no amplification product appears when other seven kinds of protozoan genomic DNA and double distilled water are used as templates; similarly, the Battrachomonas hominis species-specific primer has specificity only for Battrachomonas hominis, and can amplify a specific target band with the expected size of 623 bp (as shown in Fig. 3), while no corresponding band is amplified with other protozoan DNA and double distilled water.

[0076] (2) The results of double nested PCR specificity test

[0077] As shown in Fig. 4, using the positive controls of Pentatrichomonas hominis and Battrachomonas hominis mixed as DNA template, first using the Trichomonas-specific primer and then using the Pentatrichomonas hominis species-specific primer for amplification can amplify the expected target band with the size of 339 bp, first using the Trichomonas-specific primer and then using the Battrachomonas hominis species-specific primer for amplification can amplify the expected target band with the size of 623 bp; at the same time, using the positive controls of Pentatrichomonas hominis and Battrachomonas hominis as templates, first using the Trichomonas-specific primer and then using the mixture of the two Trichomonas species-specific primers for PCR amplification can amplify two target bands with the expected size. Using other parasitic DNA and double distilled water as templates, PCR reaction using the mixture of the two Trichomonas species-specific primers has no target band.

[0078] 4. The results of double nested PCR sensitivity test

[0079] As shown in Fig. 5, the results of double nested PCR show that after 10-fold serial dilution of 50 ng / μL positive control of Pentatrichomonas hominis and Battrachomonas hominis recombinant, PCR amplification shows that 1:10 5 diluted positive control DNA can be detected, i.e. the lowest detection sensitivity can detect 500 fg / μL.

[0080] 5. The results of stability test

[0081] Each test was repeated 3 times, and the corresponding results were successfully amplified, indicating that the established PCR method was stable.

[0082] 6. Test results of clinical samples

[0083] Table 3 Comparison of detection results of single nested PCR and double nested PCR

[0084] Of the 300 collected fresh fecal samples of cattle, 22 positive samples of Pentastomum americanum and 37 positive samples of Chlamydia psittaci were detected by single nested PCR, and 5 mixed samples of the two trichomonads were detected. The newly established double nested PCR method detected 21 positive samples of Pentastomum americanum and 37 positive samples of Chlamydia psittaci, and 5 mixed samples of the two trichomonads were detected. The results showed that the newly established double nested PCR method was highly consistent with the single nested PCR method, and there was no significant difference (P>0.05). Similarly, the double nested PCR method and the single nested PCR method also showed high consistency in detecting the infection of Pentastomum americanum and Chlamydia psittaci in pig, raccoon and deer fecal samples. In addition, the newly established double nested PCR method can significantly save the test time and reduce the test cost (Table 3).

[0085] Although preferred embodiments of the application have been described, those skilled in the art will be able to make additional changes and modifications without departing from the spirit and scope of the application. Therefore, the appended claims are intended to be interpreted as including all such changes and modifications that fall within the scope of the application.

[0086] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A PCR method for simultaneous detection of Trichomonas vaginalis and Trichomonas barbali, characterized in that, Specifically, the steps include the following: S1, First round of PCR: Using a mixture of positive control samples of Trichomonas vaginalis and Trichomonas barleyi as templates, amplification was performed using Trichomonas genus-specific primers; S2, second round of PCR: using the amplification product obtained in S1 as a template, and using a mixture of primers specific to Trichomonas vaginalis and Trichomonas barbali as primers for amplification; Two target fragments with sizes of 339bp and 623bp were obtained by amplification, indicating that both Trichomonas vaginalis and Trichomonas barleyi were detected simultaneously.

2. The dual nested PCR method for simultaneously detecting Trichomonas vaginalis and Trichomonas barbali as described in claim 1, characterized in that, In S1, the sequence of the upstream primer F of the Trichomonas-specific primer is shown in SEQ ID No. 1, and the sequence of the downstream primer R is shown in SEQ ID No.

2.

3. The dual nested PCR method for simultaneously detecting Trichomonas vaginalis and Trichomonas barbali as described in claim 1, characterized in that, In S2, the species-specific primers for Tetrachitrata barbali include the upstream primer TF with the sequence shown in SEQ ID No. 3 and the downstream primer TR with the sequence shown in SEQ ID No.

4.

4. The dual nested PCR method for simultaneously detecting Trichomonas vaginalis and Trichomonas barbali as described in claim 1, characterized in that, In S2, the human trichomonad species-specific primers include the upstream primer PF with the sequence shown in SEQ ID No. 5 and the downstream primer PR with the sequence shown in SEQ ID No.

6.

5. The dual nested PCR method for simultaneously detecting Trichomonas vaginalis and Trichomonas barbali as described in claim 1, characterized in that, In S1, the PCR reaction conditions were: 95℃ for 10 min, then 95℃ for 60 s, 60℃ for 60 s, 72℃ for 60 s, for a total of 30 cycles, and finally extended at 72℃ for 7 min.

6. The dual nested PCR method for simultaneously detecting *Trichomonas pentatrifolius* and *Tetratrias barleyi* according to claim 1, characterized in that, In S2, the PCR reaction conditions were: 95℃ for 10 min, then 95℃ for 60 s, 58℃ for 60 s, 72℃ for 60 s, for a total of 30 cycles, and finally extended at 72℃ for 7 min.

7. The dual nested PCR method for simultaneously detecting Trichomonas vaginalis and Trichomonas barbali as described in claim 1, characterized in that, In S2, the 339bp target fragment is Trichomonas vaginalis DNA.

8. The dual nested PCR method for simultaneously detecting Trichomonas vaginalis and Trichomonas barbali as described in claim 1, characterized in that, In S2, the 623bp target fragment is the DNA of Tetratrichomoniasis barbali.

9. The application of the dual nested PCR method according to any one of claims 1-8, capable of simultaneously detecting Trichomonas vaginalis and Trichomonas barleyi, in aquaculture.

10. The application according to claim 9, characterized in that, The aforementioned double nested PCR method is used to simultaneously detect Trichomonas vaginalis and Trichomonas barbatulum in the feces of multiple animal hosts such as cattle, deer, pigs, and raccoon dogs.

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