Multiplex PCR (Polymerase Chain Reaction) primer group and detection method for simultaneously detecting four kinds of animal-zoonosis protozoa of goat

Abstract

The invention discloses a multiple PCR (Polymerase Chain Reaction) primer group and a detection method for simultaneously detecting four kinds of animal-and-human protozoa of goats, which are used for detecting enterosporidium pichia, cystic protozoa, cryptosporidium parvum and giardia duodenalis. The primer group comprises the following primers: a specific primer of enterosporidium pichia, a specific primer of cyst protozoa, a specific primer of cryptosporidium parvum and a specific primer of giardia duodenalis. In addition, the invention also discloses a multiple PCR (Polymerase Chain Reaction) detection method for the enterosporidium pichia, the cystozoon, the cryptosporidium parvum and the giardia duodenalis. Experiments prove that the method has the advantages of rapidness, sensitivity and high specificity, the operation steps of traditional step-by-step PCR are remarkably reduced, the purpose of simultaneously detecting a plurality of pathogen target genes is achieved through one-time reaction, the detection cost is saved while the detection time is shortened, a foundation is laid for accurately and rapidly identifying pathogens, and the method has a relatively great development prospect.

Description

Technical Field

[0001] The present invention relates to the field of agriculture and animal quarantine technology, and in particular to a method for detecting Enterosporidium bieneusi, Blastocystis spp., Cryptosporidium parvum and Giardia duodenalis, and more particularly to a multiplex PCR primer set and a detection method for simultaneously detecting four zoonotic protozoa in goats. Background Art

[0002] Diarrhea has a high morbidity and mortality rate in both human and animal populations. The identification of common pathogens of diarrhea is of great significance for the control and prevention of such diseases. Blastocystis ), Giardia duodenalis ( Giardia duodenalis , referred to as Giardia), Cryptosporidium parvum ( Cryptosporidium parvum , Cryptosporidium) and Enterosporidiosis ( Enterocytozoon bieneusi Microsporidia (also known as microsporidia) are four common intestinal protozoa that cause diarrhea. They have a wide range of hosts, including domestic animals, wildlife, and humans. Mild infection with these protozoa can cause emaciation, anemia, slow growth, and low feed conversion rates in goats. Severe infection can lead to morbidity and even mass mortality in the herd, causing significant economic losses to the livestock industry. Furthermore, zoonotic parasitic diseases caused by parasitic protozoa in goats pose a significant threat to human health.

[0003] Cryptosporidium parvum primarily parasitizes the host's gastrointestinal epithelial cells, causing acute diarrhea in children. Currently, there is no effective treatment for cryptosporidiosis. Enterococcus bieneusi is a unicellular, obligate intracellular eukaryotic parasite. Research indicates that Enterococcus bieneusi is the most common species of microsporidia infecting humans. Giardia duodenalis primarily parasitizes the duodenum of humans and mammals, and 200 million people in Africa, Asia, and Latin America are reported to be infected with giardiasis. Blastocystis was long considered a harmless yeast, primarily parasitizing the ileocecal region of the human intestine and the intestines of cattle and sheep. Recent studies have shown that Blastocystis is a major pathogen causing chronic diarrhea. All four parasites can infect humans and animals through water, food, and other sources. Clinical symptoms vary depending on the host's immune system. Mild infections cause symptoms such as indigestion, diarrhea, and abdominal pain, while severe infections can be fatal.

[0004] The examination methods of parasitic diseases include pathogenic examination, immunological examination and molecular biological examination. The traditional pathogenic examination has higher requirements for the professional skills of the detection personnel, the precision of the detection equipment, is time-consuming and laborious, and has missed detection. The immunological diagnosis method has high sensitivity and strong specificity, but has poor test repeatability, a complex preparation process of monoclonal antibodies, low antigen yield or short storage period and the like. At present, the PCR technology gradually replaces the traditional microscope examination with the characteristics of high sensitivity, high specificity and high efficiency, and is widely applied to the detection of parasitic diseases. Chen Yaming et al. established a multiplex PCR detection method for three blood protozoa of Toxoplasma gondii, Theileria annulata and Neospora caninum, the method has strong specificity and high sensitivity, and has good application effect in the detection of 629 samples, and provides a detection method for single or mixed infection of the three blood protozoa. Chen Guoliang et al. established a multiplex PCR method for rapid detection of four kinds of pig tapeworm cysts, and the interspecific specificity is strong, and the four kinds of pig tapeworm cysts can be identified by amplification bands. The parasitic diseases of human and animals not only threaten human health, but also seriously affect the economic benefits of animal breeding industry, directly endanger the frontline breeding personnel, and may cause major public health problems. The conventional single pathogen PCR cannot meet the demand of detecting multiple parasites at the same time, and it is of great significance to establish a rapid and accurate detection method for detecting multiple parasites for the diagnosis, prevention and treatment of parasitic diseases. At present, there is no research literature on the detection of duodenal giardia, cryptosporidium parvum, blastocystis and intestinal microsporidium by multiplex PCR method. SUMMARY

[0005] One of the technical problems to be solved by the present application is to provide a multiplex PCR primer set for simultaneously detecting four zoonotic protozoa in goats.

[0006] The second technical problem to be solved by the present application is to provide a multiplex PCR reaction condition and detection method for four important protozoa (intestinal microsporidium, blastocystis, cryptosporidium parvum and duodenal giardia) in goats. The establishment of the multiplex PCR method for the four parasitic protozoa solves the cumbersome steps of multiple PCR detection, can detect multiple genes at the same time in one reaction, clearly determines the role of the pathogen, greatly reduces the workload, shortens the detection time, reduces the detection cost, and has great development prospect.

[0007] To achieve the above-mentioned purpose, the present application is implemented according to the following technical scheme: In one aspect of the present application, a multiplex PCR primer set for simultaneously detecting four zoonotic protozoa in goats is provided, which is used for simultaneously detecting intestinal microsporidium, blastocystis, cryptosporidium parvum and duodenal giardia, and the primer set comprises the following primers: Specific primer of Enterocytozoon bieneusi: F: 5'-CAAGAGTGTCTATGGTGGATGC-3', R: 5'-CGAACACTAAGATTTCTCCCGC-3', Specific primer of Cystoisospora: F: 5'-GCCCTATCAGCTTTGGATGG-3', R: 5'-GAATACCCCCAACTGTCCCT-3', Specific primer of Cryptosporidium parvum: F: 5'-GCTCTTGGACCTTGGAAAACG-3', R: 5'-GCTCGTCCATACCTTCAATCC-3', Specific primer of Giardia duodenale: F: 5'-CCTGTGCTTAAGTTCCGACG-3', R: 5'-CGGTACTGCTAGCACATTCC-3'.

[0008] Another aspect of the present application provides a multiplex PCR detection method for Enterocytozoon bieneusi, Cystoisospora, Cryptosporidium parvum and Giardia duodenale, and the specific steps include the following: (1) obtaining DNA of a sample to be detected containing Enterocytozoon bieneusi, Cystoisospora, Cryptosporidium parvum and Giardia duodenale; (2) using the DNA of the sample to be detected as a template, and using the four specific primers of claim 1 to perform multiplex PCR amplification on Enterocytozoon bieneusi, Cystoisospora, Cryptosporidium parvum and Giardia duodenale, and the multiplex PCR amplification system is 30.0 μL, wherein 2x Taq Master Mix (Dye Plus) is 15 μL, the concentration of the four specific primers is 25 μmol / L, the upper and lower stream primers are each 1 μL, ddH2O is 1 μL, and the DNA of the four samples to be detected is each 1.5 μL, and the reaction conditions are: pre-denaturation 95℃ for 5 min, denaturation 95℃ for 30 s, annealing 55℃ for 30 s, extension 72℃ for 86 s for a total of 35 cycles, and 72℃ for 10 min.

[0009] Compared with the prior art, the present application establishes a multiplex PCR detection method for simultaneously detecting Enterocytozoon bieneusi, Cystoisospora, Cryptosporidium parvum and Giardia duodenale, which can simultaneously amplify four kinds of parasite genes in one PCR reaction system, 18S rRNA, SSU rRNA, SSU rRNA, VSP with INRThe gene) not only makes up for the low detection rate, time-consuming and laborious of the morphological method, but also avoids the high false positive of the immunological method. Through test verification, the application has the advantages of rapidness, sensitivity and strong specificity, significantly reduces the operation steps of the traditional step-by-step PCR, achieves the purpose of simultaneously detecting multiple pathogenic target genes in one reaction, shortens the detection time and saves the detection cost, lays a foundation for accurate and rapid identification of pathogens, and has great development prospect. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 is a schematic diagram of plasmid identification results in embodiment 1 of the application, wherein M: 2000 bp DNA marker; N: negative control; 1: intestinal microsporidium of Bie; 2: blastocystis; 3: cryptosporidium parvum; and 4: duodenal giardia.

[0011] Figure 2 is a schematic diagram of PCR results of the multiplex PCR method for detecting four kinds of protozoa in embodiment 1 of the application, wherein M: 2000 bp DNA marker; 1: PCR products of four kinds of parasites; and N: negative control.

[0012] Figure 3 is a schematic diagram of cross-reaction (specificity) experiment of the multiplex PCR for detecting four kinds of parasites in embodiment 1 of the application, wherein M: 2000 bp DNA marker; N: negative control; 1: intestinal microsporidium of Bie; 2: blastocystis; 3: cryptosporidium parvum; and 4: duodenal giardia.

[0013] Figure 4 is a comparison of detection results of the multiplex PCR method for detecting other parasites and the four kinds of parasites mentioned in the application in embodiment 1 of the application, wherein M: 2000 bp DNA marker; 1: mixed solution of plasmids of duodenal giardia, cryptosporidium parvum, blastocystis and intestinal microsporidium of Bie; 2: DNA of intestinal microsporidium of Bie; 3: DNA of blastocystis; 4: DNA of cryptosporidium parvum; 5: DNA of duodenal giardia; 6: DNA of echinococcus granulosus; 7: DNA of proglottid disc; 8: DNA of Japanese blood fluke; 9: DNA of expanded moniezia; 10: DNA of flat stomach sucker; and N: negative control.

[0014] Figure 5 is a sensitivity test result diagram of the four kinds of parasites of the multiplex PCR method in embodiment 1 of the application, wherein M: 2000 bp DNA marker; N: negative control; 1: 1x10 6 copies / μL of parasite DNA; 2: 1x10 5 copies / μL of parasite DNA; 3: 1x10 4copies / μL; 4: Parasite DNA 1×10 3 copies / μL; 5: Parasite DNA 1×10 2 copies / μL; 6: Parasite DNA 10 copies / μL; 7: Parasite DNA 1 copies / μL; N: negative control.

[0015] Figure 6 is a schematic diagram of the detection results of the DNA extracted from the goat feces samples in Embodiment 1 of the present application, wherein M: 2000 bp DNA marker; 1-130: DNA sample multiplex PCR detection results; N: negative control. DETAILED DESCRIPTION

[0016] In order to make the objects, technical solutions and advantages of the present application clearer and more apparent, the present application will be further described in detail below in combination with embodiments. The specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0017] Embodiment 1; I. Materials and methods 1. Materials 1.1 Sample source Fresh feces samples were collected from four goat farms in Zhanjiang City, Guangdong Province.

[0018] 1.2 Parasite DNA Duodenum Giardia DNA, Cryptosporidium parvum DNA, Enterocytozoon bieneusi DNA and Cystoisospora DNA were all preserved in the laboratory.

[0019] 1.3 Plasmid construction vector and main reagents E.Z.N.A. Stool DNA Kit (D4015-02) was purchased from OMEGA Bio-Tek Company, USA; pMD18-T vector and DL2000 DNA Marker were purchased from TaKara Company; 2×Taq Master Mix (Dye Plus) and Fastpure Plasmid Mini Kit (DC201-01) were purchased from Nanjing Nvigan Biotech Co., Ltd.; GoldView nucleic acid stain (1000×) was purchased from PhyGene Company; SanPrep Column DNA Gel Extraction Kit (B518131-0100) was purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0020] 2. Methods 2.1 Primer design The gene sequences of four kinds of parasites were downloaded from GenBank, and the primer sequences and amplification fragment sizes are shown in Table 1.

[0021] Table 1: Multiplex PCR primer sequences ; 2.2 Plasmid construction The four kinds of parasite DNA stored in the laboratory were used as templates for PCR amplification of the target fragments, gel electrophoresis and recovery of the target fragments, and the recombinant plasmids of the target fragments and pMD18-T vector were constructed, wherein the PCR reaction systems of the four kinds of parasites were the same, and the extension time was different, as shown in Tables 2 and 3, wherein the cycle parameters were 35 cycles.

[0022] Table 2: Single gene PCR amplification reaction system ; Table 3: Single parasite PCR cycle parameters ; The PCR amplification products were recovered according to the instructions of the gel recovery kit, and then were ligated to the pMD18-T vector, 4 ℃ overnight, and the next day was transformed as follows: Transformation method: ① competent cells were taken out from the -80 ℃ freezer, and were thawed in ice; ② The ligation product and the competent cells were mixed at a ratio of 1:10, and were incubated in ice bath for 30 min; ③ 42 ℃ water bath for 90 s, and then ice bath for 2 min; ④ Add LB without antibiotics at a ratio of 1:20, and incubate in a shaker at 37 ℃, 220 r / min, for 1 h; ⑤ Take 1 / 10 to a culture medium containing ampicillin resistance, and evenly spread; ⑥ Incubate the culture plate in a constant temperature incubator at 37 ℃ for 12 h; pick a single colony in 5 mL of LB containing 0.15% ampicillin resistance, and incubate in a shaker at 37 ℃, 220 r / min, for 12 h; enrich the bacterial solution to 1.5 mL centrifuge tube, and extract the plasmid according to the instructions of the plasmid extraction kit.

[0023] 2.3 Specificity test The four kinds of parasite plasmid mixture were detected by single primer, and the DNA of B. intestinalis, B. cysticercus, C. parvum, D. duodenal, J. japonicum, P. ovale, E. granulosus, B. cysticercus and P. phlyctenium were detected by multiplex PCR method to evaluate the specificity of the method.

[0024] 2.4 Sensitivity test The concentration of the recombinant plasmid was measured respectively, the plasmid concentration was converted into copy number, and was diluted to 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , and 10 copies / μL respectively, and the mixed solution of different concentrations of plasmids was detected by multiplex PCR to evaluate the sensitivity.

[0025] Table 4: Four kinds of parasite DNA copy number ; Plasmid copy number (copies / μL) = concentration (ng / μL) × 6.023 × 10 14 / (base number × 660); After dilution, the copy number was 1 × 10 6 copies / μL, 1 × 10 5 copies / μL, 1 × 10 4 copies / μL, 1 × 10 3 copies / μL, 1 × 10 2 copies / μL, 10 copies / μL, and 1 copy / μL, respectively.

[0026] 2.5 Application of multiplex RCR The multiplex PCR method was applied to detect 130 fecal DNA samples, and the detection results were compared with the single pathogen PCR detection results of the DNA of the four kinds of parasites.

[0027] II. Results 1. Plasmid construction The constructed plasmids were identified by PCR, and it can be seen from Figure 1 that the target fragments with the expected size were obtained.

[0028] 2. Establishment of multiplex PCR detection method According to the instructions of 2×Taq Master Mix (Dye Plus), the PCR system, annealing temperature and other conditions were explored; the established multiplex PCR system was 30.0 μL, including 2×Taq Master Mix (Dye Plus) 15.0 μL, 1.0 μL of each of the upstream and downstream primers (25 μmol / L), 1.0 μL of ddH2O and 1.5 μL of DNA template. Reaction conditions: pre-denaturation 95 ℃ 5 min; denaturation 95 ℃ 30 s; annealing 55 ℃ 30 s; extension 72 ℃ 86 s for 35 cycles; 72 ℃ 10 min.

[0029] The four goat parasitic protozoan genomic DNAs were mixed and subjected to PCR amplification using the established multiplex PCR detection method, and the results are shown in Figure 2 , which showed good amplification effect, proving that the method can simultaneously detect the four goat zoonotic parasitic protozoans of P. biechleri, B. samirami, C. parvum and J. duodenalis.

[0030] 3. Specificity test The method has good specificity to the genes of the parasitic protozoan group, can amplify specific bands, and does not appear cross amplification between the four parasitic protozoans, and the results are shown in Figure 3 , each pair of primers can amplify specific bands of the target genes, and will not amplify other bands.

[0031] No amplification bands were observed for T. tenuis, P. posthunnum, S. japonicum, M. expansa and P. phlyctenium, and the results are shown in Figure 4 , the four pairs of primers only produce specific amplification bands for the target genes, and will not amplify specific bands for the genomic DNAs of other parasitic protozoans.

[0032] 4. Sensitivity test As shown in Figure 5 , the detection limit of the J. duodenalis primer in the multiplex PCR method is ≥ 10 2 copies / μL, the detection limit of the C. parvum primer is ≥ 10 2 copies / μL, the detection limit of the B. samirami primer is ≥ 10 2 copies / μL, and the detection limit of the P. biechleri primer is ≥ 10 copies / μL. The detection limit of the multiplex PCR method is ≥ 10 2 copies / μL of parasitic protozoan DNA clone plasmid.

[0033] 5. Multiplex PCR detection results of goat fecal samples The laboratory collected goat fecal samples were detected using the above established multiplex PCR method, and the results are shown in Figure 6 , among the 130 goat fecal DNA samples, the number of backup goat fecal DNA samples was 64, of which 44 (44 / 64, 68.75%) fecal sample DNAs were positive by single pathogen PCR detection; the number of adult goat samples was 66, of which 36 (36 / 66, 54.54%) fecal sample DNAs were positive by single pathogen PCR detection. The single pathogen PCR detection results of the two groups of positive DNA samples were consistent with the established multiplex PCR detection results.

[0034] The technical solution of the present application is not limited to the above specific embodiments, and any technical modification made according to the technical solution of the present application falls within the protection scope of the present application.

Claims

1. A multiplex PCR primer set for simultaneous detection of four human-animal shared parasitic protozoa in goats, characterized by, It is used for detecting simultaneously Enterocytozoon bieneusi, Blastocystis sp., Cryptosporidium parvum and Giardia duodenalis, and the primer set comprises the following primers: Specific primers for Enterocytozoon bieneusi: F: 5'-CAAGAGTGTCTATGGTGGATGC-3', R: 5'-CGAACACTAAGATTTCTCCCGC-3', Specific primers for Blastocystis sp.: F: 5'-GCCCTATCAGCTTTGGATGG-3', R: 5'-GAATACCCCCAACTGTCCCT-3', Specific primers for Cryptosporidium parvum: F: 5'-GCTCTTGGACCTTGGAAAACG-3', R: 5'-GCTCGTCCATACCTTCAATCC-3', Specific primers for Giardia duodenalis: F: 5'-CCTGTGCTTAAGTTCCGACG-3', R: 5'-CGGTACTGCTAGCACATTCC-3'.

2. A multiplex PCR detection method of Enterocytozoon bieneusi, Cystoisospora, Cryptosporidium parvum and Giardia duodenalis, characterized in that, The specific steps comprise the following: (1) obtaining DNA of samples containing Enterocytozoon bieneusi, Blastocystis sp., Cryptosporidium parvum and Giardia duodenalis to be detected; (2) using the DNA of the detection samples as a template, the four specific primers of claim 1 are used to carry out multiplex PCR amplification on Enterocytozoon bieneusi, Blastocystis sp., Cryptosporidium parvum and Giardia duodenalis, and the multiplex PCR amplification system is 30.0 μL, wherein 2x Taq Master Mix (Dye Plus) is 15 μL, the concentration of the four specific primers is 25 μmol / L, the upstream and downstream primers are each 1 μL, ddH2O is 1 μL, and the DNA of the four samples to be detected is each 1.5 μL, the reaction conditions are as follows: pre-denaturation 95 ℃ 5 min; denaturation 95 ℃ 30 s; annealing 55 ℃ 30 s; extension 72 ℃ 86 s for a total of 35 cycles; 72 ℃, 10 min.

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