Duplex PCR method for detecting entamoeba suis and entamoeba polecki and use thereof

Abstract

The present invention relates to the technical field of molecular detection, and aims to provide a primer set for a duplex PCR method for simultaneously detecting Entamoeba suis and Entamoeba polecki, a method, and a use. The primer set comprises the following primers: species-specific primers for Entamoeba suis, namely forward primer E.suis F and reverse primer E.suis R, and species-specific primers for Entamoeba polecki, namely forward primer E.polecki F and reverse primer E.polecki R. The nucleotide sequences of E.suis F, E.suis R, E.polecki F, and E.polecki R are respectively shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4. A duplex PCR detection system having high specificity, high sensitivity, time- and labor-saving properties, and easy operation is established. The primer combination and method can synchronously detect Entamoeba suis and Entamoeba polecki in a single reaction, can significantly reduce the cost and workload of detecting the two pathogens, and can detect positive DNA at a minimum concentration of 400 fg / μL. The method can be applied to the rapid detection and identification of infections caused by these two protozoa in the porcine intestinal tract.

Description

Double PCR detection method for E. suis and E. polecki and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular detection, and particularly relates to a double PCR method for simultaneously detecting E. suis and E. polecki. BACKGROUND

[0002] Amoeba is a zoonotic protozoan that can parasitize the intestinal tract and other internal organs of humans and animals, and has many species. According to the living environment, the amoeba is divided into two types, i.e., an internal amoeba and a free-living amoeba. The internal amoeba parasitizes humans and animals, and mainly includes four genera, i.e., Entamoeba, Endolimax, Iodamoeba and Dientamoeba. Among them, Entamoeba histolytica (E. histolytica) in the genus Entamoeba is the only internal amoeba protozoan with pathogenicity, which can cause 50 million people to suffer from amebic colitis and liver abscesses every year, and cause about 100,000 people to die, and has become one of the important pathogens that endanger global public health.

[0003] At present, the reported internal amoeba species that can infect pigs mainly include E. suis and E. polecki. Although it has been reported that pigs can be infected with E. histolytica under experimental conditions, so far, it has not been found that pigs can be naturally infected with the protozoan. Studies have shown that E. suis mainly infects pigs, and can invade the intestinal mucosa of pigs to cause hemorrhagic colitis. Unlike E. suis that only infects pigs, E. polecki can infect a variety of hosts including humans, non-human primates, other mammals and birds. Traditionally, E. polecki is considered to be non-pathogenic, but recent studies have shown that when the protozoan co-infects with other pig intestinal pathogens, it can aggravate the proliferative enteritis of pigs. Therefore, it is necessary to further clarify the potential role of pigs in the zoonotic transmission of internal amoeba, and therefore, it is crucial to develop a detection method for internal amoeba.

[0004] In clinic, pigs infected with Entamoeba usually have no or mild symptoms, and the diagnosis must rely on laboratory diagnosis. At present, the methods for detecting Entamoeba mainly include microscopic examination, culture method and molecular detection method. Microscopic examination and culture method mainly involve direct microscopic examination of feces or microscopic examination after culture of feces, and rely on observation of the morphology of Entamoeba cysts or trophozoites for judgment. However, the morphologies of cysts or trophozoites of different Entamoeba species are extremely similar, and it is difficult to distinguish them under a light microscope, which can easily cause false positive results. Therefore, at present, species identification and genotyping of Entamoeba are mainly carried out by molecular diagnostic methods. At present, some molecular methods for detecting Entamoeba have been developed, but the related research mainly focuses on E. histolytica, and there are few molecular methods for other Entamoeba species. Moreover, most of the related methods can only detect one species at a time, which is time-consuming, laborious and costly.

[0005] SUMMARY

[0006] To solve the above technical problems, the present application provides a double PCR method for simultaneously detecting porcine Entamoeba and Entamoeba polecki, which specifically amplifies the target fragments of porcine Entamoeba and Entamoeba polecki, and has no cross reaction with the genomic DNA of 10 common intestinal parasites of pigs such as Cryptosporidium, Giardia lamblia, Blastocystis, Enterocytozoon bieneusi, Balantidium coli, Isospora suis, Trichuris suis, Ascaris suum, Trichonema suis and Strongylus suis. The minimum positive control DNA that can be detected is 400 fg / μL.

[0007] A primer set for double PCR for simultaneously detecting porcine Entamoeba and Entamoeba polecki, comprising the following primers:

[0008] The upstream primer E.suis F and the downstream primer E.suis R of the porcine Entamoeba species-specific primer, and the upstream primer E.polecki F and the downstream primer E.polecki R of the Entamoeba polecki species-specific primer;

[0009] The nucleotide sequences of E.suis F, E.suis R, E.polecki F and E.polecki R are shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0010] A method for double PCR of the primer set for simultaneously detecting porcine Entamoeba and Entamoeba polecki, comprising the following steps:

[0011] Using the genomic DNA of the sample to be detected as a template, and using the porcine Entamoeba species-specific primer and the Entamoeba polecki species-specific primer as amplification primers for PCR amplification, and electrophoresis;

[0012] If electrophoresis obtains two target fragments with sizes of 320 bp and 600 bp, Entamoeba suis and Entamoeba polecki are detected simultaneously.

[0013] Preferably, the reaction conditions of the PCR are as follows: 95℃ for 4 min, then 95℃ for 45 s, 54℃ for 30 s, 72℃ for 45 s, for 35 cycles, and finally 72℃ for 7 min.

[0014] Preferably, the target fragment with the size of 320 bp is Entamoeba suis DNA.

[0015] Preferably, the Entamoeba suis DNA is Entamoeba suis SSU rDNA, and the nucleotide sequence is shown as SEQ ID No. 5.

[0016] Preferably, the target fragment with the size of 600 bp is Entamoeba polecki DNA.

[0017] Preferably, the Entamoeba polecki DNA is Entamoeba polecki SSU rDNA, and the nucleotide sequence is shown as SEQ ID No. 6.

[0018] The application of the double-PCR method for simultaneously detecting Entamoeba suis and Entamoeba polecki to simultaneously detecting Entamoeba suis and Entamoeba polecki in pig feces.

[0019] Preferably, the method is used for detecting Entamoeba suis and Entamoeba polecki in pigs.

[0020] Compared with the prior art, the application has the beneficial effects that:

[0021] 1. The double-PCR method can specifically amplify the target fragments of Entamoeba suis and Entamoeba polecki, and has no cross reaction with the genomic DNA of 10 common intestinal parasites in pigs, such as Cryptosporidium parvum, Giardia lamblia, Blastocystis hominis, Enterocytozoon bieneusi, Balantidium coli, Isospora suis, Trichuris suis, Ascaris suum, Trichomonas suis and Strongylus ransomi, and can detect 400 fg / μL of positive control DNA at the lowest;

[0022] 2. The double-PCR method provided by the application is rapid, accurate, simple, and can be directly used for simultaneously detecting Entamoeba suis and Entamoeba polecki in pig feces in the clinic, and can be used for rapid detection and identification of the two parasites, and has important significance for epidemiological investigation and prevention and control of the two parasites in pig farms;

[0023] 3. The double-PCR method has high specificity, and the PCR amplification can detect 1:104 The diluted positive control DNA, i.e. the lowest detectable sensitivity is 400 fg / μL, the detection results of the double-PCR method are consistent with those of the single-PCR method, and the consistency is significantly different (P>0.05), and the double-PCR method of the application can significantly save test time and reduce test cost. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0025] Fig. 1 is the specific test result of pig entamoeba in the embodiment of the present application;

[0026] Among them, the meaning of each lane is as follows: M: DL2000 DNA Marker; 1: pig entamoeba; 2: cryptosporidium; 3: giardia lamblia; 4: blastocystis; 5: intestinal microsporidium; 6: balloon ciliates; 7: isospora suis; 8: trichuris suis; 9: ascaris suum; 10: trichuris suis; 11: strongyloides ransomi; 12: double distilled water;

[0027] Fig. 2 is the specific test result of entamoeba hartmanni in the embodiment of the present application;

[0028] Among them, the meaning of each lane is as follows: M: DL2000 DNA Marker; 1: entamoeba hartmanni; 2: cryptosporidium; 3: giardia lamblia; 4: blastocystis; 5: intestinal microsporidium; 6: balloon ciliates; 7: isospora suis; 8: trichuris suis; 9: ascaris suum; 10: trichuris suis; 11: strongyloides ransomi; 12: double distilled water;

[0029] Fig. 3 is the specific test result of double-PCR product amplification in the embodiment of the present application;

[0030] Among them, the meaning of each lane is as follows: M: DL2000 DNA Marker; 1: entamoeba hartmanni; 2: pig entamoeba; 3: pig entamoeba+entamoeba hartmanni; 4: cryptosporidium; 5: giardia lamblia; 6: blastocystis; 7: intestinal microsporidium; 8: balloon ciliates; 9: isospora suis; 10: trichuris suis; 11: ascaris suum; 12: double distilled water;

[0031] Fig. 4 is the sensitivity test result of double-PCR in the embodiment of the present application;

[0032] In which, each lane meaning as follows: M: DL2000 DNA Marker; 1: 1-9 are positive templates, 1-9 respectively represent 1:10 0 -1:10 8 Dilution, 10: blank control. DETAILED DESCRIPTION

[0033] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts are within the scope of protection of the present application. The experimental methods described in each embodiment of the present application are conventional methods unless otherwise specified.

[0034] EMBODIMENT

[0035] I. Materials and methods

[0036] 1. Materials

[0037] (1) DNA of insect strain: Genomic DNA of common intestinal parasites of pigs, such as Cryptosporidium parvum, Giardia lamblia, Blastocystis sp., Enterocytozoon bieneusi, Balantidium coli, Isospora suis, Trichuris suis, Ascaris suum, Trichomonas suis and Strongylus sp., were isolated and preserved by the Animal Medical Experimental Teaching Center of the Animal Science College of Anhui Science and Technology University. The positive controls of Entamoeba suis and Entamoeba polecki were recombinant plasmid DNA obtained by inserting the SSU rDNA fragments of Entamoeba suis and Entamoeba polecki from pigs into pMD18-T vector, with a concentration of 4 ng / μL, prepared and preserved by the laboratory. The sequence of SSU rDNA of Entamoeba suis is shown in SEQ ID No. 5, and the sequence of SSU rDNA of Entamoeba polecki is shown in SEQ ID No. 6.

[0038] (2) Collection of clinical detection fecal samples: A total of 410 fresh pig fecal samples were collected from multiple large-scale pig farms, with about 100 g of each sample packed into clean plastic bags and taken back to the laboratory for detection at 4°C in a refrigerator.

[0039] (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 Tian Gen Biochemical Technology (Beijing) Co., Ltd.

[0040] 2. Methods

[0041] (1) Design and synthesis of primers

[0042] Table 1 PCR primers used in this example

[0043] The primers were designed according to the SSU rDNA genes of Entamoeba porci and Entamoeba boti, as shown in Table 1, and were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0044] (2) PCR amplification

[0045] (2.1) Entamoeba porci PCR

[0046] The reaction system is shown in Table 2. The PCR used a positive control of Entamoeba porci as a template, and the primers used were the Entamoeba porci species-specific primers in Table 1.

[0047] (2.2) Entamoeba boti PCR

[0048] The reaction system is shown in Table 2. The PCR used a positive control of Entamoeba boti as a template, and the primers used were the Entamoeba boti species-specific primers in Table 1.

[0049] (2.3) Duplex PCR

[0050] The reaction system is shown in Table 2. The PCR template used a mixture of positive controls of Entamoeba porci and Entamoeba boti, and the primers used were a mixture of the Entamoeba porci species-specific primers and the Entamoeba boti species-specific primers in Table 1.

[0051] (2.4) PCR reaction conditions

[0052] Regardless of single PCR or duplex PCR, the PCR reaction conditions were 95°C for 4 min, then 95°C for 45 s, 54°C for 30 s, 72°C for 45 s, for a total of 35 cycles, and finally 72°C for 7 min.

[0053] Table 2 PCR reaction system

[0054] (2.5) Detection

[0055] After the reaction, a 1.5% agarose gel was prepared by adding EB, 20 μL of the PCR product and 2 μL of 10x electrophoresis loading buffer were mixed and then loaded, and electrophoresis was carried out at a voltage of 120 V. When the bromophenol blue was about 1 cm from the other end of the gel, the electrophoresis was stopped, and the gel was observed under a gel imaging system, photographed, and the results were recorded.

[0056] (3) PCR reaction specificity test

[0057] (3.1) Single PCR specificity test

[0058] Using positive control DNA from *Entamoeba hepatica* and *Entamoeba bortani* as templates, and genomic DNA from other protozoa such as *Cryptosporidium*, *Giardia lamblia*, *Baumannii*, *Microsporidium bifidum*, *Balticidium coli*, *Isospora suis*, *Trichomonas suis*, *Ascaris suis*, *Trichuris suis*, and *Strongyloides suis*, as well as double-distilled water as templates, PCR was performed using *Entamoeba hepatica* species-specific primers or *Entamoeba bortani* specific primers to detect the specificity of single-item PCR.

[0059] (3.2) Dual PCR Specificity Assay

[0060] Using positive control DNA from *Entamoeba hepatica* and *Entamoeba bortani* as templates, and genomic DNA from *Cryptosporidium*, *Giardia lamblia*, *Baumannii*, *Microsporidium bifidum*, *Balticidium coli*, *Isospora suis*, *Trichomonas suis*, and *Ascaris suis*, along with double-distilled water as templates, PCR was performed using *Entamoeba hepatica* species-specific primers and *Entamoeba bortani* species-specific primers, alone or in combination, to identify their specificity.

[0061] (4) Sensitivity test of dual PCR reaction

[0062] Dilute to a starting concentration of 4 ng / μL, and then use a 10-fold dilution method, successively diluting 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 1:10 8 Serial dilutions were performed, and the positive control DNA of *Entamoeba porcini* and *Entamoeba borteri* was diluted to nine concentration gradients. 1 μL of each dilution was taken as a PCR template for duplex PCR amplification. The amplification products were subjected to 1.5% agarose gel electrophoresis to detect their sensitivity.

[0063] (5) Stability test

[0064] Each experiment was repeated three times to verify the stability of the experimental method.

[0065] (6) Testing of clinical samples

[0066] Genomic DNA was extracted from 410 fresh pig fecal samples using a fecal DNA extraction kit. The DNA was then detected using single PCR and a dual PCR method for detecting Entamoeba histolytica and Entamoeba borzei, respectively. The experimental results were compared.

[0067] II. Results and analysis

[0068] 1. PCR specificity test results

[0069] (1) Single PCR specificity test results

[0070] As shown in Figure 1, using the Entamoeba suis single PCR method, amplification results showed that only the Entamoeba suis positive control as a template amplified a specific band of the expected size, 320 bp, and no amplification product was present for the other 10 pig-derived parasite genomic DNA and double distilled water (as shown in Figure 1). Similarly, the Entamoeba polecki species-specific primer was only specific to Entamoeba polecki, amplifying a specific band of the expected size, 600 bp, while no corresponding band was amplified for other pig-derived parasite DNA and double distilled water (as shown in Figure 2).

[0071] (2) Duplex PCR specificity test results

[0072] As shown in Figure 3, using the Entamoeba suis and Entamoeba polecki positive controls mixed as DNA templates, amplification with the Entamoeba suis species-specific primer alone could amplify the expected band of 320 bp, amplification with the Entamoeba polecki species-specific primer alone could amplify the expected band of 600 bp, and amplification with the mixture of the Entamoeba suis and Entamoeba polecki species-specific primers could amplify two bands of the expected size. Using other parasite DNA and double distilled water as templates, PCR amplification with the mixture of the Entamoeba suis and Entamoeba polecki species-specific primers did not produce the expected bands (as shown in Figure 3).

[0073] 2. Duplex PCR sensitivity test results

[0074] As shown in Figure 4, the duplex PCR results showed that after 10-fold serial dilution of 4 ng / μL Entamoeba suis and Entamoeba polecki recombinant positive control, PCR amplification could detect 1:10 4 diluted positive control DNA, i.e., the lowest detectable sensitivity was 400 fg / μL.

[0075] 3. Stability test results

[0076] Each test was repeated three times, and the corresponding results were successfully amplified, indicating that the established PCR method had good stability.

[0077] 4. Test results of clinical samples

[0078] Table 3 comparison of detection results of single PCR and duplex PCR

[0079] For the 410 collected fresh pig fecal samples, 25 samples of Entamoeba suis and 40 samples of Entamoeba polecki were detected positive by single PCR, and 10 samples were mixed infection samples of Entamoeba suis and Entamoeba polecki, 23 samples of Entamoeba suis and 40 samples of Entamoeba polecki were detected positive by the newly established duplex PCR, and 10 samples were mixed infection samples, which showed that the detection results of the newly established duplex PCR and single PCR were consistent, and there was no significant difference (P>0.05), and the newly established duplex PCR could significantly save the test time and reduce the test cost (Table 3).

[0080] It should be noted that when the present application claims involving numerical ranges, it should be understood that each numerical range of two endpoints and any number between the two endpoints can be selected, and in order to prevent repetition, the present application describes the preferred embodiments.

[0081] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0082] 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 equivalents, the present application also intends to include these modifications and variations.

Claims

1. A primer set for duplex PCR for simultaneous detection of Entamoeba porci and Entamoeba botini, characterized by, The primers comprise the following: An upstream primer E.suis F and a downstream primer E.suis R of the Entamoeba suis species-specific primer, an upstream primer E.polecki F and a downstream primer E.polecki R of the Entamoeba polecki species-specific primer; The nucleotide sequences of the E.suis F, E.suis R, E.polecki F, and E.polecki R are shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively.

2. The method of claim 1, wherein the double PCR for simultaneously detecting Entamoeba polecki and Entamoeba porci is characterized by, The method comprises the following steps: PCR amplification is performed using the genomic DNA of the sample to be tested as a template and the Entamoeba suis species-specific primer and the Entamoeba polecki species-specific primer as amplification primers, and electrophoresis is performed; If two target fragments with sizes of 320 bp and 600 bp are obtained by electrophoresis, it is determined that both the Entamoeba suis and the Entamoeba polecki are detected.

3. The method of the duplex PCR for simultaneously detecting Entamoeba porci and Entamoeba botini according to claim 2, characterized in that, The reaction conditions of the PCR are as follows: 95℃ for 4 min, then 95℃ for 45 s, 54℃ for 30 s, 72℃ for 45 s, for a total of 35 cycles, and finally 72℃ for 7 min.

4. The method of the duplex PCR for simultaneously detecting Entamoeba porci and Entamoeba botini according to claim 2, characterized in that, The target fragment with a size of 320 bp is the Entamoeba suis DNA.

5. The method of the duplex PCR for simultaneously detecting Entamoeba porci and Entamoeba botini according to claim 4, characterized in that, The Entamoeba suis DNA is the SSU rDNA of the Entamoeba suis, and the nucleotide sequence is shown in SEQ ID No.

5.

6. The method of the duplex PCR for simultaneously detecting Entamoeba porci and Entamoeba botini according to claim 2, characterized in that, The target fragment with a size of 600 bp is the Entamoeba polecki DNA.

7. The method of the duplex PCR for simultaneously detecting Entamoeba porci and Entamoeba botini according to claim 6, characterized by, The Entamoeba polecki DNA is the SSU rDNA of the Entamoeba polecki, and the nucleotide sequence is shown in SEQ ID No.

6.

8. The method of any one of claims 2-7 for simultaneous detection of the Entamoeba suis and the Entamoeba polecki in the intestinal tract of a pig.

9. Use according to claim 8, characterized in that, The method is used for detection of the Entamoeba suis and the Entamoeba polecki infection in the intestinal tract of a pig.

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