Synchronous detection method for enterosporidium pichia and cystozoon in complex water body and application of synchronous detection method for enterosporidium pichia and cystozoon pichia in complex water body

Abstract

The invention belongs to the technical field of environmental monitoring and biological detection, and discloses a synchronous detection method for enterosporidium pichia and cystoprotozoa in a complex water body and application, the method comprises the following steps: S1, pretreatment of a water body sample; s2, nucleic acid extraction; s3, carrying out PCR (Polymerase Chain Reaction) detection; based on a specially designed specific primer, specific nested PCR amplification, agarose gel electrophoresis imaging, gene bidirectional sequencing and sequence alignment are performed on an ITS gene of the enterosporidium pichia and a 500bp SSUrRNA gene of the cystoprotozoa, and then whether a sample contains the enterosporidium pichia and the cystoprotozoa is judged according to an electrophoresis result. And finally, identifying the genotypes / new genotypes of the enterosporidium pichia and the cystoprotozoa through gene sequencing. Compared with traditional observation under a microscope and other methods, the method is higher in efficiency and sensitivity, can quickly identify insect species and genotypes / new genotypes thereof, and is wide in application range.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental monitoring and biological detection, and particularly relates to a method for synchronous detection of Enterocytozoon bieneusi and Blastocystis in complex water bodies and application thereof. BACKGROUND

[0002] Enterocytozoon bieneusi and Blastocystis are important zoonotic opportunistic pathogenic intestinal protozoa, which parasitize in the intestinal epithelial cells of humans and various animals. More than 90% of microsporidiosis is caused by Enterocytozoon bieneusi. After human infection with Enterocytozoon bieneusi, different symptoms such as asymptomatic, chronic diarrhea, abdominal pain, fever, nausea, malnutrition, etc. are presented due to individual differences. Infection of low immune human groups can cause fatal diarrhea. After human infection with Blastocystis, the symptoms and severity presented are related to the individual immunity. For normal immune human groups, it may only show self-limiting diarrhea or no obvious symptoms, while for low immune human groups (such as HIV patients, transplant patients and dialysis patients), it may cause clinical syndromes mainly with diarrhea. Enterocytozoon bieneusi and Blastocystis are widely distributed in the environment, and can be transmitted through water and food, etc. to cause outbreak of epidemic, which seriously endangers public health safety.

[0003] Since the individual of Enterocytozoon bieneusi and Blastocystis is very small, after fluorescent staining marking, there is a high risk of missed detection by microscopy, and the fuel is easily decomposed by light, which is not conducive to long-term preservation of the sample. Although the enzyme-linked immunosorbent assay (ELISA) has high specificity, sensitivity, stability and repeatability, the detection cost of this method is high, and it is not suitable for large-scale sample detection. PCR technology has high specificity and high sensitivity, and has been widely used in the detection of various pathogenic organisms. However, the PCR method in the prior art still has problems such as incomplete removal of interference of complex water samples or missed detection, low detection efficiency and sensitivity, and inability to detect and identify genotypes / subtypes and conduct environmental risk assessment.

[0004] For example, the Chinese invention patent application document CN 111269999 A discloses a universal primer, kit, test strip and application for simultaneously detecting four kinds of human microsporidia, which constructs a PCR reaction system containing sample DNA and universal primer, performs gel electrophoresis after PCR reaction, and judges whether the microsporidia are contained according to the electrophoresis result, thereby improving the specificity of PCR detection, but the amplification specific primer and reaction system cannot be directly applied to the synchronous detection of B. intestinalis and blastocystis in complex water samples, and the detection efficiency is low, the sensitivity is not high, and the genotype / new genotype of the sample cannot be identified, which cannot meet the efficient detection of complex water samples and further expand the application. In addition, the prior art also has the problems of false positive results due to poor primer specificity, indirectly leading to increased workload of sequencing and verification, detection cost waste, cost increase and the like.

[0005] Therefore, the prior art cannot meet the needs of large-scale, wide-range, low-cost environmental monitoring, public health assessment and clinical diagnosis. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide a synchronous detection method and application of B. intestinalis and blastocystis in complex water bodies, which is based on specially designed specific primers, and through the screening of specific primers of B. intestinalis and blastocystis in complex water bodies and the optimization of PCR reaction system, the specificity of the primers is optimized, false positives are avoided, the detection cost is reduced, the genotype / new genotype is identified, and the risk of zoonosis and the potential harm to public health are evaluated according to phylogenetic and genetic evolution analysis, which can meet the needs of large-scale, wide-range, low-cost environmental monitoring, public health assessment and clinical diagnosis.

[0007] The technical scheme provided by the present application to solve the above problems is:

[0008] A synchronous detection method of B. intestinalis and blastocystis in complex water bodies, characterized in that it comprises the following steps:

[0009] S1: Pretreatment of water sample

[0010] The method of vacuum filtration, acetone dissolution filter membrane, addition of PBS buffer solution, centrifugation and collection of precipitate is used to enrich microorganisms in complex water samples, and remove impurities such as humic acid, metal ions, proteins, polysaccharides, salts and the like that may interfere with the PCR detection result;

[0011] S2: Nucleic acid extraction

[0012] The precipitate is vortexed with buffer solution until fully mixed, proteinase is added and centrifuged to obtain supernatant, and the whole genome DNA of the sample is extracted by using a nucleic acid extraction kit.

[0013] S3: PCR detection

[0014] The ITS gene site-specific primer is used for PCR amplification of Enterocytozoon bienei, and the 500bp SSUrRNA gene site-specific primer is used for PCR amplification of Blastocystis, and the positive product is sequenced in two directions to qualitatively determine whether Enterocytozoon bienei and Blastocystis exist in the water sample.

[0015] S4: Genotype / subtype identification: The positive product of the two-worm PCR amplification is sequenced in two directions, and BLAST comparison is performed with the known sequences in the GenBank database to further determine whether it is Enterocytozoon bienei and Blastocystis, identify the genotype / subtype, and find whether there is a new genotype.

[0016] S5: Environmental risk assessment

[0017] The genotype, phylogeny and genetic evolution of Enterocytozoon bienei and Blastocystis in the sample are analyzed to assess whether the two worms have the risk of spreading in humans, animals and the environment and the potential harm to public health.

[0018] The application of the synchronous detection method of Enterocytozoon bienei and Blastocystis in the complex water body is applied to the assessment of whether the two worms have the risk of spreading in humans, animals and the environment and the potential harm to public health according to the phylogeny and genetic evolution analysis of Enterocytozoon bienei and Blastocystis; it can also be applied to the rapid identification of Enterocytozoon bienei and Blastocystis, molecular diagnosis of related intestinal infectious diseases, tracing and prevention and control.

[0019] The synchronous detection method of Enterocytozoon bienei and Blastocystis in the complex water body provided by the application and the application have at least the following beneficial effects compared with the prior art:

[0020] 1. The PCR method provided by the application has high specificity and sensitivity, can completely avoid false positives, is not dependent on human subjective factors, has relatively low cost, can simultaneously process samples in batches, and can accurately identify the genotype / new genotype of Enterocytozoon bienei and Blastocystis on the basis of evaluating the zoonotic risk of the two intestinal protozoa, and can provide scientific basis for the tracing of the two intestinal protozoa, rapid diagnosis and effective prevention and control of related intestinal infectious diseases, and is a research hotspot in the field at present.

[0021] 2. The application provides a molecular biology synchronous detection method for Enterocytozoon bieneusi and Cystoisospora, based on specially designed specific primers, and has high detection efficiency and sensitivity.

[0022] 3. The application further compares the amplification effects of two specific primers of the nested PCR, wherein one is a specially designed and improved primer of the application, and the primer amplification and sequencing effects have obvious advantages; the improved Enterocytozoon bieneusi ITS gene site amplification primer has higher specificity than the original document (avoiding false positives), but has the same specific amplification effect as the 16s rRNA gene site, and the ITS amplification and sequencing results can be directly used for genotyping of Enterocytozoon bieneusi, which is beneficial to the discovery of new genotypes and the tracing and effective prevention and control of pathogens. The improved Cystoisospora 500bp SSU rRNA gene site amplification primer has higher specificity, effectively avoids false positive results, improves the accuracy of sequencing, and reduces the detection cost.

[0023] 4. The application uses a sterile mixed cellulose ester filter membrane with a pore size of 0.22 microns to extract complex water samples, which can capture most microorganisms in the complex water body except viruses; and through the pretreatment steps of acetone dissolution and concentration, the capture efficiency of Enterocytozoon bieneusi and Cystoisospora is improved. The application uses a high-power diaphragm vacuum pump for vacuum filtration, which improves the filtration efficiency of complex water samples.

[0024] 5. The PCR amplification primers used in the application have high specificity and sensitivity, and reduce the missed detection rate of Enterocytozoon bieneusi and Cystoisospora.

[0025] 6. Through actual testing, the detection method and application provided by the application have the advantages of rapid, efficient, convenient batch detection process, accurate detection results, breaking through the limitations of existing similar products and technologies, and meeting the needs of large-scale, wide-range, low-cost popularization and application and popularization. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a gel electrophoresis diagram of PCR amplification of four Enterocytozoon bieneusi positive animal feces nucleic acid samples and four Enterocytozoon bieneusi negative animal feces nucleic acid samples identified by the application as DNA templates, using the Enterocytozoon bieneusi ITS gene specific primer (A) and the 16s rRNA gene specific primer (B) of the application.

[0027] Figure 2 is a gel electrophoresis diagram of PCR products of the orthogonal experiment of two rounds of PCR amplification of different annealing temperatures using the identified 3 pieces of Enterocytozoon bieneusi positive animal fecal nucleic acid sample DNA templates in the embodiment 1 of the present application.

[0028] Figure 3 is a gel electrophoresis diagram of PCR amplification products of the comparative experiment of different annealing temperatures using the identified 3 pieces of Cystoisospora positive animal fecal nucleic acid sample DNA templates in the embodiment 2 of the present application.

[0029] Figure 4 Figure 4 is a gel electrophoresis diagram of PCR products of the PCR amplification of the identified 3 pieces of Enterocytozoon bieneusi positive animal fecal nucleic acid sample DNA templates using two different specific primers of the ITS gene site of Enterocytozoon bieneusi before and after optimization in the embodiment 3 of the present application.

[0030] Figure 5 Figure 5 is a gel electrophoresis diagram of PCR products of the PCR amplification of the identified 3 pieces of Cystoisospora positive animal fecal nucleic acid sample DNA templates using two different specific primers of the 500bp SSUrRNA gene site of Cystoisospora before and after optimization in the embodiment 4 of the present application.

[0031] Figure 6 Figure 6 is a gel electrophoresis diagram of the sensitivity detection of the PCR amplification of the identified 3 pieces of Enterocytozoon bieneusi positive animal fecal nucleic acid sample DNA templates after dilution by different multiples using the specific primers of the ITS gene site of Enterocytozoon bieneusi in the embodiment 5 of the present application.

[0032] Figure 7 Figure 7 is a gel electrophoresis diagram of the sensitivity detection of the PCR amplification of the identified 3 pieces of Cystoisospora positive animal fecal nucleic acid sample DNA templates after dilution by different multiples using the specific primers of the 500bp SSUrRNA gene site of Cystoisospora in the embodiment 6 of the present application.

[0033] Figure 8 is a gel electrophoresis diagram of the PCR amplification of the identified 4 pieces of Cystoisospora positive animal fecal nucleic acid samples and 4 pieces of Cystoisospora negative animal fecal nucleic acid samples using the specific primers of the 260bp SSUrRNA gene of Cystoisospora (A) and the specific primers of the 500bp SSUrRNA gene (B) in the embodiment 7 of the present application.

[0034] Figure 9 Figure 9 is a gel electrophoresis diagram of the PCR detection of the water samples of the rural surface sewage channel using the specific primers of the ITS gene of Enterocytozoon bieneusi in the embodiment 10 of the present application.

[0035] Figure 10The gel electrophoresis diagram of PCR detection of water samples of rural surface sewage channels using the specific primer pair of the 500bp SSUrRNA gene of Blastocystis for Example 10 of the present application. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions of the present application are further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0037] The instruments involved in the following examples are: diaphragm vacuum pump (GM-2, Tianjin Teng Experimental Equipment Co., Ltd., Tianjin, China), table-top refrigerated centrifuge (5424R, Eppendorf Co., Ltd., Germany), vortex shaker (MX-S, SCILOGEX Co., Ltd., USA), biosafety level II operating table (Thermo Fisher Co., Ltd., USA), tissue disruptor (DS1000, Hubei Xinyongke Virus Disease Engineering Technology Co., Ltd., China), low-temperature linker instrument (Hema L1, Zhuhai Heima Medical Instrument Co., Ltd., China), table-top high-speed small centrifuge (5415R, Eppendorf Co., Ltd., Germany), mini microcentrifuge (MiniSpin Plus, Eppendorf Co., Ltd., Germany), gradient PCR amplifier (nexus SX1, Eppendorf Co., Ltd., Germany), electrophoresis instrument (Wide Mini-sub Cell GT, Bio-Rad Co., Ltd., USA), chemical light / fluorescence image analysis system (Tanon 5200 Multi, Shanghai Tian Neng Life Science Co., Ltd., China).

[0038] The reagent consumables involved in the following examples are: Amicrom sterile mixed cellulose ester filter membrane with a diameter of 50mm and a pore size of 0.22μm (Hangzhou Micron Pa Technology Co., Ltd., China), QIAamp Fast DNA Stool Mini kit reagent box (Qiagen Co., Ltd., Germany), 6×Loading Buffer (Takara Co., Ltd., Japan), dNTP (Takara Co., Ltd., Japan), Taq DNA polymerase (Takara Co., Ltd., Japan), DEPC (Thermo Fisher Co., Ltd., USA), 50×TAE (Biosharp Co., Ltd., Hefei, China), 3%6×Super GelRed (Taian Xinzhongsheng Biotechnology Co., Ltd., China), DL2000Marker (Takara Co., Ltd., Japan). TM (China, Taian Xinzhongsheng Biotechnology Co., Ltd.), DL2000Marker (Takara Co., Ltd., Japan).​

[0039] The total volume of the PCR reaction system involved in the following examples is 25 μL, containing 2 μL of sample DNA as a template, 0.5 μL of specific PCR amplification primers for the forward primer and the reverse primer, respectively.

[0040] The PCR reaction product is detected by 1.5% agarose gel electrophoresis.

[0041] Basic examples

[0042] The synchronous detection method for Enterocytozoon bieneusi and Cystoisospora in complex water bodies provided in the present examples focuses on improving the specificity of the detection method, and comprises the following steps:

[0043] S1: Pretreatment of water sample

[0044] The microorganisms in the complex water sample are enriched by the method of vacuum filtration, acetone-dissolved filter membrane, addition of PBS buffer, and collection of the precipitate after centrifugation, and the impurities such as humic acid, metal ions, proteins, polysaccharides, and salts that may interfere with the PCR detection results are removed;

[0045] S2: Nucleic acid extraction

[0046] The precipitate is vortexed with the buffer until it is fully mixed, and the supernatant is collected after centrifugation after the addition of protease, and the whole genome DNA of the sample is extracted using a nucleic acid extraction kit;

[0047] S3: PCR detection

[0048] The ITS gene site-specific primer is used for PCR amplification of Enterocytozoon bieneusi, and the 500 bp SSUrRNA gene site-specific primer is used for PCR amplification of Cystoisospora, and the positive product is bidirectionally sequenced to qualitatively determine whether Enterocytozoon bieneusi and Cystoisospora exist in the water sample.

[0049] S4: Genotype / subtype identification: The positive product of the PCR amplification of the two worms is bidirectionally sequenced, and BLAST comparison is performed with the known sequences in the GenBank database to further determine whether it is Enterocytozoon bieneusi and Cystoisospora, identify the genotype / subtype, and find out whether there is a new genotype.

[0050] S5: Environmental risk assessment

[0051] The genotypes, phylogeny, and genetic evolution of Enterocytozoon bieneusi and Cystoisospora in the sample are analyzed to assess whether the two worms have the risk of spreading in humans, animals, and the environment and the potential harm to public health.

[0052] The application of the method for synchronous detection of Enterocytozoon bieneusi and Cystoisospora in complex water bodies is applied to evaluate whether the two parasites have the risk of spreading in humans, animals and the environment and the potential harm to public health, and can also be applied to rapid identification of Enterocytozoon bieneusi and Cystoisospora, molecular diagnosis of related intestinal infectious diseases, tracing, and prevention and control.

[0053] Embodiment 1

[0054] The method for synchronous detection of Enterocytozoon bieneusi and Cystoisospora in complex water bodies and the application thereof provided by the application are specific applications based on the foregoing embodiments, and the specificity of primers in detection effect is improved by optimizing the primers, so as to solve the problems of the prior art, such as false positive results caused by slightly poor specificity of primers, indirectly leading to increased workload of sequencing and verification, detection fund waste, cost increase, and the like. The specificity of the optimized primers is improved in this embodiment, false positives are avoided, and detection costs are reduced.

[0055] Based on the annealing temperature range of the nested PCR amplification reaction, the orthogonal experiment of different annealing temperatures is designed for the specific primers of the nested PCR amplification of the ITS gene site of Enterocytozoon bieneusi in the application (as shown in Table 1), and the reaction program is as shown in Table 2, and the orthogonal experiment design of the annealing temperature is as shown in Table 3. Using the identified 3 positive animal fecal nucleic acid sample DNA templates of Enterocytozoon bieneusi, the reaction system is prepared according to Table 4, and the PCR amplification experiment of different annealing temperatures is carried out to optimize the reaction program.

[0056] Table 1

[0057]

[0058] Table 2

[0059]

[0060] Table 3

[0061] Serial number First round annealing temperature Second round annealing temperature Gel electrophoresis number 1 51℃ 52℃ 1-3 2 51℃ 55℃ 4-6 3 54℃ 52℃ 7-9 4 54℃ 55℃ 10-12 5 57℃ 55℃ 13-15 6 57℃ 52℃ 16-18 7 57℃ 58℃ 19-21 8 54℃ 58℃ 22-24 9 51℃ 58℃ 25-27

[0062] Table 4

[0063] Name Volume (μL) 10 x Buffer 2.5 dNTP (10 mM) 2 Primer F (10 μM) 0.5 Primer R (10 μM) 0.5 Taq DNA polymerase (5 U / μL) 0.5 Template DNA 2 DEPC water 17 Total 25

[0064] The nested PCR amplification reaction program of the ITS gene site of Enterocytozoon bieneusi in the embodiment of the application is optimized, and the experimental results show that the optimal annealing temperature of the first round of reaction of the specific primer nested PCR amplification of the ITS gene site of Enterocytozoon bieneusi is 57℃, and the optimal annealing temperature of the second round of reaction is 55℃, as shown in Figs. 2(A) and 2(B).

[0065] Example 2

[0066] The method and application for simultaneous detection of *Microsporidium pichii* and *Blastocystis* in complex water bodies provided by this invention are specific applications based on the aforementioned embodiments. Based on the annealing temperature range of nested PCR amplification reactions, comparative experiments were designed with different annealing temperatures (52℃, 54℃, 56℃, 58℃) ​​targeting the specific primers for nested PCR amplification of the 500bp SSUrRNA gene locus of *Blastocystis* in this invention (as shown in Table 5). The reaction procedures are shown in Table 6. Using three identified *Blastocystis*-positive animal fecal nucleic acid samples as DNA templates, the reaction system was prepared according to Table 7, and PCR amplification experiments were conducted at different annealing temperatures to optimize the reaction procedure.

[0067] Table 5

[0068]

[0069] Table 6

[0070]

[0071] Table 7

[0072] Name Volume (μL) 10 x Buffer 2.5 dNTP (10 mM) 2 Primer F (10 μM) 0.5 Primer R (10 μM) 0.5 Taq DNA polymerase (5 U / μL) 0.5 Template DNA 2 DEPC water 17 Total 25

[0073] This invention optimizes the nested PCR amplification procedure for the 500bp SSUrRNA gene locus of *Blastocystis*, and the experimental results ( Figure 3A (B) indicates that the optimal annealing temperature for nested PCR amplification of the 500bp SSUrRNA gene locus specific to *Blastocystis* was 54℃, yielding the best gel electrophoresis imaging results. As shown in Figure 3, gel electrophoresis samples 1-3 were annealed at 54℃, 4-6 at 52℃, 7-9 at 56℃, and 10-12 at 58℃.

[0074] Example 3

[0075] The method and application for simultaneous detection of *P. pylori* and *Blastocystis* in complex water bodies provided by this invention are specific applications based on the aforementioned embodiments. Specifically, this invention optimizes the specific primers for nested PCR amplification of the *P. pylori* ITS gene locus by adding two bases to the upstream and downstream 3' ends of the primers. To verify the specificity of the optimized primers, this invention uses four animal fecal nucleic acid samples already identified as *P. pylori* positive and four animal fecal nucleic acid samples already identified as *P. pylori* negative as DNA templates. Nested PCR amplification is performed on these eight samples using primers before and after optimization. The reaction system is shown in Table 8, and the reaction procedure is shown in Table 9.

[0076] Table 8

[0077]

[0078]

[0079] Table 9

[0080]

[0081] Referring to Figure 4 Fig. 1-8 are gel electrophoresis imaging of the nested PCR amplification products of the optimized primers (i.e. two bases are added to the 3' end of the upstream and downstream of the primers, respectively), wherein 1-4 are identified negative samples of Enterocytozoon bieneusi, 5-8 are identified positive samples of Enterocytozoon bieneusi; 9-16 are gel electrophoresis imaging of the nested PCR amplification products of the primers before optimization, wherein 9-12 are identified negative samples of Enterocytozoon bieneusi, 13-16 are identified positive samples of Enterocytozoon bieneusi, and false positive appears in the amplification of sample 12. The experimental results show that the nested PCR amplification using the optimized Enterocytozoon bieneusi ITS gene site-specific primers in the present application has stronger specificity, more accurate and reliable amplification and gel electrophoresis imaging, can effectively avoid false positive, and reduce detection cost.

[0082] Example 4

[0083] The method for simultaneous detection of Enterocytozoon bieneusi and Coccidium in complex water bodies and the application thereof provided by the embodiments of the present application are specific applications based on the foregoing embodiments, and further optimize the 500bp SSUrRNA gene site-specific amplification primers of Coccidium by adding two bases to the 3' end of the upstream and downstream of the primers, respectively. In order to test the specificity of the optimized primers, the present application uses 4 animal fecal nucleic acid sample DNA templates identified as positive for Coccidium and 4 animal fecal nucleic acid samples identified as negative for Coccidium as DNA templates, and performs nested PCR amplification on the 8 samples using the primers before and after optimization, respectively. The reaction system is shown in Table 10, and the reaction procedure is shown in Table 11.

[0084] Table 10

[0085] Name Volume (μL) 10 x Buffer 2.5 dNTP (10 mM) 2 Primer F (10 μM) 0.5 Primer R (10 μM) 0.5 Taq DNA polymerase (5 U / μL) 0.5 Template DNA 2 DEPC water 17 Total 25

[0086] Table 11

[0087]

[0088] Referring to Figure 5, and 8 are the gel electrophoresis imaging of the nested PCR amplification products of the optimized primers (i.e. adding two bases respectively at the 3' end of the upstream and downstream of the primers), wherein 1-4 are the identified positive samples of Cystoisospora, and 5-8 are the identified negative samples of Cystoisospora; 9-16 are the gel electrophoresis imaging of the nested PCR amplification products of the primers before optimization, wherein 9-12 are the identified positive samples of Cystoisospora, and 13-16 are the identified negative samples of Cystoisospora, and the samples of 14 and 16 appear false positive in amplification. The experimental results show that the nested PCR amplification primers of the 500bp SSUrRNA gene site of Cystoisospora in the application have no false positive experimental results, the specificity is obviously improved, false positive can be completely avoided, and the experimental results are more accurate and reliable.

[0089] Example 5

[0090] The simultaneous detection method and application of the Enterocytozoon bieneusi and Cystoisospora in the complex water body provided by the embodiments of the application are specific applications based on the foregoing embodiments. In order to test the sensitivity of the specific primer nested PCR amplification of the ITS gene site of Enterocytozoon bieneusi in the application, 3 positive animal fecal nucleic acid sample DNA templates of Enterocytozoon bieneusi identified are used, and the sample nucleic acid concentrations obtained by Nanodrop determination are shown in Table 12. The undiluted DNA templates, the 3-fold diluted DNA templates and the 10-fold diluted DNA templates of the 3 positive samples are amplified by the specific primers of the ITS gene site of Enterocytozoon bieneusi in the application, the reaction system is shown in Table 13, and the reaction procedure is shown in Table 14.

[0091] The electrophoresis results are shown in Table 15, wherein 1-3 are the nested PCR amplification products of the undiluted DNA templates, 4-6 are the nested PCR amplification products of the 3-fold diluted DNA templates, and 7-9 are the nested PCR amplification products of the 10-fold diluted DNA templates. The experimental results show that the specific primers of the ITS gene site of Enterocytozoon bieneusi in the application have high sensitivity. Figure 6

[0092] Table 12

[0093]

[0094] Table 13

[0095] Name Volume (μL) 10 x Buffer 2.5 dNTP (10 mM) 2 Primer F (10 μM) 0.5 Primer R (10 μM) 0.5 Taq DNA polymerase (5 U / μL) 0.5 Template DNA 2 DEPC water 17 Total 25

[0096] Table 14

[0097]

[0098]

[0099] Example 6​

[0100] The application embodiment provides the complex water body in the synchronous detection method and application of the intestinal microsporidian of the Bie and the blastocystis, which is a specific application based on the foregoing embodiment. In order to test the sensitivity of the 500bp SSUrRNA gene site-specific primer nested PCR amplification of the blastocystis in the application, 3 positive animal fecal nucleic acid sample DNA templates identified by the blastocystis are used. The sample nucleic acid concentration obtained by Nanodrop determination is shown in Table 15. The undiluted DNA template, the 3-fold diluted DNA template and the 10-fold diluted DNA template of the 3 positive samples are amplified by the specific primer of the 500bp SSUrRNA gene site of the blastocystis in the application, the reaction system is shown in Table 16, and the reaction procedure is shown in Table 17.

[0101] The electrophoresis result is shown in Table 18. Figure 7 The 1-3 are the 10-fold diluted DNA template nested PCR amplification products, the 4-6 are the undiluted DNA template nested PCR amplification products, and the 7-9 are the 3-fold diluted DNA template nested PCR amplification products. The experimental results show that the specific primer of the 500bp SSUrRNA gene site of the blastocystis in the application has high sensitivity.

[0102] Table 15

[0103]

[0104] Table 16

[0105]

[0106]

[0107] Table 17

[0108]

[0109] Example 7

[0110] Referring to FIG. 8, the application embodiment provides the complex water body in the synchronous detection method and application of the intestinal microsporidian of the Bie and the blastocystis, which is basically the same as the embodiments 1 to 6, and the difference is that:

[0111] The screening of the blastocystis PCR amplification specific primer includes the following contents:

[0112] Using the identified 4 samples of coccidium positive animal feces nucleic acid (No. 1, No. 6, No. 7, No. 8 samples) and 4 samples of coccidium negative animal feces nucleic acid (No. 2-5 samples) as DNA templates, PCR detection was carried out using coccidium 260bp SSUrRNA gene site-specific primer and the optimized 500bp SSUrRNA gene site-specific primer of the application, respectively. The gel electrophoresis diagram of the amplification product is shown in Fig. 8 (A) and Fig. 8 (B).

[0113] The coccidium 260bp SSUrRNA gene site-specific amplification PCR reaction program is as follows:

[0114] 94℃ denaturation for 5min; 94℃ denaturation for 1min, 53℃ annealing for 1min, 72℃ extension for 1min, a total of 35 cycles; finally 72℃ extension for 10min. 4℃∞.

[0115] The coccidium 500bp SSUrRNA gene site-specific amplification PCR reaction program is as follows:

[0116] 95℃ denaturation for 4min; 95℃ denaturation for 30s, 54℃ annealing for 30s, 72℃ extension for 30s, a total of 35 cycles; finally 72℃ extension for 5min. 4℃∞.

[0117] As shown in Fig. 8, Fig. 8 is the gel electrophoresis diagram of PCR amplification using the coccidium 260bp SSUrRNA gene specific primer (A) and 500bp SSUrRNA gene specific primer (B). The results show that the specificity of PCR amplification using coccidium 260bp SSUrRNA gene site-specific primer is low, and false positive results (No. 2 sample is false positive) are prone to occur. The specificity of PCR detection of 500bp SSUrRNA gene site using the optimized specific primer of the application is stronger, and the accuracy and gene bidirectional sequencing success rate are higher.

[0118] Example 8

[0119] The synchronous detection method of Entamoeba histolytica and coccidium in complex water body provided by the embodiment comprises the following steps:

[0120] S1: Pretreatment of water sample

[0121] Vacuum filtration, acetone dissolution of filter membrane, addition of PBS buffer solution, and centrifugal collection of precipitate are used to enrich microorganisms in complex water samples and remove impurities that may interfere with PCR detection results;

[0122] The filter membrane used for suction filtration is a sterile mixed cellulose ester filter membrane with a diameter of 50 mm and a pore size of 0.22 μm, which is used to enrich microorganisms in complex water samples and intercept all microorganisms in the water body except viruses, and remove interfering impurities; acetone is an analytical pure reagent.

[0123] S2: nucleic acid extraction

[0124] The QIAamp Fast DNA Stool Mini kit was used to extract the whole genome DNA from the sample, and the precipitate was vortexed with buffer until fully mixed. After adding protease, the supernatant was obtained by centrifugation, and the whole genome DNA of the sample was extracted by nucleic acid extraction kit, which included the following steps:

[0125] S2-1: Transfer the thawed precipitate sample to a 2 mL centrifuge tube, add 1 mL of Buffer, vortex until fully mixed. Then, place the sample in a metal bath at 95℃ for 5 min, vortex for 15 s, and centrifuge at 14000 r / min for 1 min at 4℃.

[0126] S2-2: Take 1.5 mL of sterile centrifuge tube, add 15 μL of protease K, 200 μL of centrifuged sample supernatant, and 200 μL of AL buffer in order, vortex for 15 s, and then place the mixed liquid sample in a metal bath at 70℃ for 10 min.

[0127] S2-3: Add 200 μL of anhydrous ethanol, vortex to mix, then add the mixed solution to the filter column, and place the filter column in a new 2 mL collection tube. Label the filter column cover, and centrifuge at 14000 r / min for 1 min at 4℃.

[0128] S2-4: Take out the centrifuged filter column, transfer it to a new 2 mL collection tube, add 500 μL of AW1 buffer, and centrifuge for the second time at 14000 r / min for 1 min at 4℃.

[0129] S2-5: Take out the centrifuged filter column again, place it in a new 2 mL collection tube, add 500 μL of AW2 buffer, and centrifuge for the third time at 14000 r / min for 1 min at 4℃.

[0130] S2-6: Transfer the centrifuged filter column to a new 2 mL collection tube, and centrifuge for the fourth time at 14000 r / min for 3 min at 4℃.

[0131] S2-7: Finally, the filter column was taken out and transferred to a new 1.5 mL centrifuge tube, 200 μL of ATE buffer was directly added, incubated at room temperature for 1 min, centrifuged at 4°C at 14000 r / min for 2 min, and the eluted DNA was stored at -80°C for standby.

[0132] S3: PCR detection

[0133] The ITS gene site-specific primer was used for PCR amplification of Enterocytozoon bienei, and the 500 bp SSUrRNA gene site-specific primer was used for PCR amplification of Blastocystis, and the positive products were bidirectional sequencing to qualitatively determine whether Enterocytozoon bienei and Blastocystis existed in the water sample.

[0134] The specific primer was used for PCR amplification of Enterocytozoon bienei and Blastocystis, and the positive products were bidirectional sequencing to qualitatively determine whether Enterocytozoon bienei and Blastocystis existed in the water sample. The Enterocytozoon bienei positive sample and Blastocystis positive sample identified by gene bidirectional sequencing were used as positive controls, and DEPC water was used as negative control. The PCR reaction system contained: 2 μL of whole genome DNA of the sample as template, 0.5 μL of specific amplification forward primer and reverse primer, 0.5 μL of 5 U / μL Taq DNA polymerase, 2.5 μL of 10×Buffer, 2 μL of 10 mM dNTP, 17 μL of DEPC water, and the total volume was 25 μL. Specifically including the following steps:

[0135] S3-1: PCR detection of Enterocytozoon bienei ITS gene site

[0136] The specific primer for the first round of PCR amplification reaction of Enterocytozoon bienei was: the PCR amplification forward primer was 5'-GGTCATAGGGATGAAGAG-3', the nucleotide sequence was as shown in SEQ ID NO. 1, and the reverse primer was 5'-TTCGAGTTCTTTCGCGCTC-3', the nucleotide sequence was as shown in SEQ ID NO. 2;

[0137] The first round of PCR amplification reaction program was: 95°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 57°C annealing for 30 s, 72°C extension for 40 s, a total of 35 cycles; finally 72°C extension for 10 min;

[0138] The specific primers for the second round of PCR amplification reaction of the intestinal microsporidium of bie's are: the PCR amplification forward primer is 5'-GCTCTGAATATCTATGGCT-3', the nucleotide sequence is shown as SEQ ID NO. 3, and the reverse primer is 5'-ATCGCCGACGGATCCAAGTG-3', the nucleotide sequence is shown as SEQ ID NO. 4;

[0139] The second round of PCR amplification reaction program is: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 40s, a total of 35 cycles; finally 72℃ extension for 10min.

[0140] S3-2: PCR detection of 500bp SSUrRNA gene site of blastocystis

[0141] The specific primers of 500bp SSU rRNA gene site are: the forward primer is 5'-GGAGGTAGTGACAATAAATC-3', the nucleotide sequence is shown as SEQ ID NO. 9, and the reverse primer is 5'-TGCTTTCGCACTTGTTCATC-3', the nucleotide sequence is shown as SEQ ID NO. 10;

[0142] The PCR amplification reaction program of blastocystis is: 95℃ denaturation for 4min; 95℃ denaturation for 30s, 54℃ annealing for 30s, 72℃ extension for 30s, a total of 35 cycles; finally 72℃ extension for 5min; 4℃∞.

[0143] Among them, the screening of PCR amplification specific primers of intestinal microsporidium of bie's

[0144] Using the identified 4 intestinal microsporidium of bie's positive animal feces nucleic acid samples and 4 intestinal microsporidium of bie's negative animal feces nucleic acid samples as DNA templates, the specific primers of ITS gene site and 16s rRNA gene site of intestinal microsporidium of bie's were detected by PCR method respectively, and the gel electrophoresis diagram of the amplification product is shown in Figure 1(A) and Figure 1(B).

[0145] The specific amplification PCR reaction program of ITS gene site of intestinal microsporidium of bie's is as follows:

[0146] First amplification: 95℃ denaturation for 3min; 94℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension for 40s, a total of 35 cycles; finally 72℃ extension for 10min; 4℃∞.

[0147] Second amplification: 94℃ denaturation for 3min; 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 40s, a total of 35 cycles; finally 72℃ extension for 10min; 4℃∞.

[0148] The PCR reaction program for site-specific amplification of the 16s rRNA gene of Enterocytozoon bieneusi is as follows:

[0149] The first and second round amplification reaction programs are consistent: denaturation at 95℃ for 5min; denaturation at 95℃ for 45s, annealing at 55℃ for 45s, extension at 72℃ for 1min, a total of 40 cycles; final extension at 72℃ for 10min; 4℃∞.

[0150] The results show that the effect of site-specific PCR amplification of the ITS gene of Enterocytozoon bieneusi is consistent with that of the 16s rRNA gene site-specific PCR amplification, but the amplified product of the ITS gene site can be sequenced to identify the genotype of Enterocytozoon bieneusi. This not only can evaluate the zoonotic risk of Enterocytozoon bieneusi in samples, but also helps to find new genotypes of Enterocytozoon bieneusi.

[0151] S4: Genotype / subtype identification: The positive product of two-worm PCR amplification was subjected to bidirectional sequencing of gene sequence, and BLAST comparison with known sequences in the GenBank database was performed to further determine whether it was Enterocytozoon bieneusi and Blastocystis, identify its genotype / subtype, and find whether there was a new genotype. The specific steps are as follows:

[0152] S4-1: The positive product of two-worm PCR amplification was subjected to bidirectional sequencing of gene sequence, and BLAST comparison with known sequences in the GenBank database was performed to further determine whether it was Enterocytozoon bieneusi and Blastocystis, and identify the genotype of Enterocytozoon bieneusi and the subtype of Blastocystis.

[0153] S4-2: According to the provisions of Enterocytozoon bieneusi genotyping, the sequence of 243 nucleotides in the ITS region of Enterocytozoon bieneusi detected in the sample had one base different from all known sequences in the GenBank database, which was identified as a new genotype of Enterocytozoon bieneusi. According to the identification provisions of new subtypes of Blastocystis, the new subtype should be based on ≥80% of CA bases in the 1800bp SSU nucleotide sequence, which had a difference of ≥4% from all known sequences in the GenBank database and was not in the same branch as all known subtypes in the phylogenetic tree, which was identified as a new subtype of Blastocystis.

[0154] S5: Environmental risk assessment

[0155] The genotype, phylogeny, and genetic evolution of Enterocytozoon bieneusi and Blastocystis in the sample were analyzed to assess the risk of transmission of Enterocytozoon bieneusi and Blastocystis in humans, animals, and the environment and the potential harm to public health, which specifically includes the following steps:

[0156] S5-1: Constructing phylogenetic tree using MEGA 11 software: importing sequence data, selecting Kimura 2-paramete as parameter model, constructing tree by Neighbor-Joining method, setting the number of Bootstrap Replications to 1000 to evaluate the reliability of the tree, and exporting the result as an image.

[0157] S5-2: Retrieving literature database, if the identified Enterocytozoon bieneusi and Cystoisospora in the sample have been reported to be detected in human samples, it indicates that the subtype has the risk of spreading to humans and will pose potential harm to public health.

[0158] The application of the method for synchronous detection of Enterocytozoon bieneusi and Cystoisospora in complex water bodies is based on phylogenetic and genetic evolution analysis of Enterocytozoon bieneusi and Cystoisospora, and is applied to evaluate the risk of transmission of Enterocytozoon bieneusi and Cystoisospora in humans, animals and the environment and the potential harm to public health. Specifically, based on the phylogenetic and genetic evolution analysis of Enterocytozoon bieneusi and Cystoisospora, if the genotype of Enterocytozoon bieneusi identified in the sample belongs to Group 1 zoonotic group in the phylogenetic tree or has been reported to be detected in human samples, it indicates that the genotype has the risk of transmission in humans, animals and the environment and the potential harm to public health; if the subtype of Cystoisospora identified in the sample has been reported to be detected in human samples, it indicates that the subtype has the risk of spreading to humans and will pose potential harm to public health.

[0159] The synchronous detection method can also be applied to the tracing of the two intestinal protozoa Enterocytozoon bieneusi and Cystoisospora, the rapid diagnosis and prevention of enteric infectious diseases.

[0160] Example 9

[0161] Referring to FIG. 1, the method for synchronous detection of Enterocytozoon bieneusi and Cystoisospora in complex water bodies provided by the embodiment of the application is basically the same as that of Example 1, and further specifically includes the following contents:

[0162] The method for synchronous detection of Enterocytozoon bieneusi and Cystoisospora in complex water bodies further includes the following steps:

[0163] (1) Sample pretreatment

[0164] The diaphragm vacuum pump and the sterile mixed cellulose ester filter membrane with a diameter of 50mm and a pore size of 0.22μm are used to vacuum filter the complex water samples (including the complex water samples containing various impurities collected directly in the field) such as surface water, underground water, drinking water, domestic sewage, aquaculture wastewater and urban greening water. The obtained filter membrane is cut into pieces with sterilized surgical scissors and placed into a 10mL sterile centrifuge tube with a cover. Then, the filter membrane is dissolved with acetone. After the filter membrane is completely dissolved, it is mixed well by vortex oscillation at 4℃ and 3000r / min for 10min. The supernatant is carefully discarded. The above operation is repeated for 3 times. Then, the PBS buffer is added into the obtained precipitate, which is mixed well by vortex oscillation at 4℃ and 3000r / min for 10min. The supernatant is carefully discarded, and the precipitate is stored at -80℃ for standby.

[0165] In the example, the 0.22μm sterile mixed cellulose ester filter membrane is used for vacuum filtration, which can intercept all microorganisms in the water body except viruses, and avoid the subsequent missed detection as much as possible in the pretreatment of the water body. At the same time, the substances interfering with the subsequent detection results are filtered out, i.e. the impurities possibly interfering with the PCR detection results are removed.

[0166] The acetone used in the example is AR grade analytical pure reagent, which has high solvent purity and very few impurities, thereby reducing the interference of solvent impurities on the subsequent experiments. The analytical pure acetone has high purity and few impurities, thereby reducing the interference of organic reagent impurities on the later molecular biology detection.

[0167] (2) Nucleic acid extraction

[0168] The QIAamp Fast DNA Stool Mini kit (produced by Qiagen Company, Germany) is used to extract the whole genome DNA in the sample, and the collective steps are as follows:

[0169] a. The thawed precipitate sample is transferred into a 2mL centrifuge tube, 1mL of Buffer is added, and vortex oscillation is performed until the mixture is uniformly mixed. Then, the sample is heated in a metal bath at 95℃ for 5min, and vortex oscillation is performed for 15s. The sample is centrifuged at 4℃ and 14000r / min for 1min.

[0170] b. A 1.5mL sterile centrifuge tube is taken, and 15μL of protease K, 200μL of the centrifuged sample supernatant and 200μL of AL buffer are sequentially added. After vortex oscillation for 15s, the mixed liquid sample is heated in a metal bath at 70℃ for 10min.

[0171] ​c. Add 200 μL of absolute ethanol, vortex to mix, and then add the mixture to the filter column. Place the filter column in a new 2 mL collection tube, label the filter column cap, and centrifuge at 14,000 r / min for 1 min at 4℃;

[0172] d. Take out the centrifuged filter column, transfer it to a new 2 mL collection tube, add 500 μL of AW1 buffer, and centrifuge for the second time at 14,000 r / min for 1 min at 4℃;

[0173] e. Take out the centrifuged filter column again, place it in a new 2 mL collection tube, add 500 μL of AW2 buffer, and centrifuge for the third time at 14,000 r / min for 1 min at 4℃;

[0174] f. Transfer the centrifuged filter column to a new 2 mL collection tube, and centrifuge for the fourth time at 14,000 r / min for 3 min at 4℃;

[0175] g. Finally, take out the filter column, transfer it to a new 1.5 mL centrifuge tube, directly add 200 μL of ATE buffer, incubate at room temperature for 1 min, centrifuge at 14,000 r / min for 2 min at 4℃, elute the DNA, and store it at -80℃ for later use.

[0176] (3) Screening of specific primers

[0177] The core technical problem to be solved by the embodiments of the present application is to simultaneously improve the detection efficiency and sensitivity of the molecular biology synchronous detection method for Enterocytozoon bieneusi and Cystoisospora. The embodiments of the present application are to compare the PCR amplification and gene sequencing results of the ITS gene site and the 16s rRNA gene site of Enterocytozoon bieneusi, compare the PCR amplification and gene sequencing results of the 500bp SSUrRNA gene site and the 260bp SSUrRNA gene site of Cystoisospora, so as to realize the screening of the specific primers for the nested PCR amplification of Enterocytozoon bieneusi and Cystoisospora and the optimization of the reaction system.

[0178] On the basis of the literature primers of Enterocytozoon bieneusi (the original material milk Enterocytozoon bieneusi ITS gene site specificity amplification primer designed by Yeliz Yildirim et al.) and the literature primers of Cystoisospora (the 500bp SSUrRNA gene site specificity amplification primer of human and animal Cystoisospora designed by Mónica Santín et al.), two bases are added to the upper and lower 3' ends, respectively, to improve the specificity of PCR amplification of the two worms.

[0179] The embodiment adopts nested PCR amplification, and a specific primer is newly and uniquely designed, bases at 3' ends of the upstream and downstream primers are increased, so that the specificity, accuracy and detection efficiency of PCR amplification are improved, and the false positive detection rate is reduced.

[0180] The embodiment adopts the nested PCR method to amplify the ITS gene site and the 16s rRNA gene site of the Enterocytozoon bieneusi, and the 500bp SSUrRNA gene site and the 260bp SSU rRNA gene site of the Blastocystis.

[0181] The embodiment compares the specific primer amplification effects of two kinds of nested PCR, one of which is a primer specially designed and improved in the embodiment, and the primer amplification and sequencing effects have obvious advantages; the improved Enterocytozoon bieneusi ITS gene site amplification primer has higher specificity than the original literature, but the sensitivity is consistent with the specific amplification effect of the 16s rRNA gene site, the ITS amplification and sequencing results can be directly used for genotype identification of Enterocytozoon bieneusi, which is conducive to the discovery of new genotypes and the tracing and effective prevention and control of pathogens. The improved 500bp SSUrRNA gene site amplification primer of Blastocystis has higher specificity, effectively avoids false positive results, improves the accuracy and efficiency of sequencing, and thus reduces the detection cost.

[0182] The primers used in the embodiment are synthesized by Shengong Biotechnology Co., Ltd. (China Shanghai), and the sequences are shown in Table 18 (specific primers of Enterocytozoon bieneusi) and Table 19 (specific primers of Blastocystis); the PCR reaction system is shown in Table 20; and the reaction conditions are shown in Table 21 (PCR reaction conditions of Enterocytozoon bieneusi) and Table 22 (PCR reaction conditions of Blastocystis).

[0183] Table 18

[0184]

[0185]

[0186] Table 19

[0187]

[0188] Table 20

[0189] Name Volume (μL) 10 x Buffer 2.5 dNTP (10 mM) 2 Primer F (10 μM) 0.5 Primer R (10 μM) 0.5 Taq DNA polymerase (5 U / μL) 0.5 Template DNA 2 DEPC water 17 Total 25

[0190] Table 21

[0191]

[0192] Table 22

[0193]

[0194]

[0195] 2 PCR amplification products were positive in 1.5% agarose gel electrophoresis imaging. The PCR amplification products were sequenced in both directions using walking method with PCR primers. The sequencing results were spliced using ClustalX 1.83, the bases were corrected according to the sequencing map, and then the corrected nucleotide sequences were BLAST compared with known sequences in the GenBank database (http: / / www.ncbi.nlm.nih.gov) to further determine whether they were Enterocytozoon bieneusi and Cystoisospora.

[0196] In this embodiment, 1.5% agarose gel was used; the sample loading amount was 6 μL, including 5 μL of PCR amplification product and 1 μL of 6xLoading Buffer; the electrophoresis instrument was set at a voltage of 130 V for 30 min.

[0197] Please refer to FIG. 1. Four Enterocytozoon bieneusi positive animal fecal nucleic acid samples and four Enterocytozoon bieneusi negative animal fecal nucleic acid samples identified in this embodiment of the application were used as DNA templates. FIG. 1(A) is a gel electrophoresis diagram of PCR amplification using the Enterocytozoon bieneusi ITS gene specific primer of the application; and FIG. 1(B) is a gel electrophoresis diagram of PCR amplification using the Enterocytozoon bieneusi 16s rRNA gene specific primer of the application.

[0198] It can be observed that the PCR amplification and gel electrophoresis results of the Enterocytozoon bieneusi ITS gene specific primer of this embodiment of the application are consistent with the PCR amplification effect of the 16s rRNA gene specific primer, but the sequencing of the ITS gene amplification product can not only determine whether it is Enterocytozoon bieneusi, but also determine the genotype of Enterocytozoon bieneusi, and assess the zoonotic risk of Enterocytozoon bieneusi in the sample according to the genotype, which is also helpful for the discovery of new genotypes of Enterocytozoon bieneusi.

[0199] The amplification efficiency of the 260 bp SSUrRNA gene site of Cystoisospora in this embodiment of the application is higher than that of the 500 bp SSUrRNA gene site, but the gene sequencing success rate of the positive PCR amplification product of the latter is much higher than that of the former.

[0200] (5) Phylogenetic analysis

[0201] The MEGA 11 software was used to construct a phylogenetic evolution tree (Bootstrap-1000) based on the Kimura two-parameter model and the Neighbor-Joining (NJ) method.

[0202] Example 10

[0203] Referring to the accompanying Figure 9 and Figure 10 , the embodiment of the application provides the synchronous detection method and application of the Enterocytozoon bieneusi and the coccidium in the complex water body, which are basically the same as those in the examples 1-3. Specifically, the Enterocytozoon bieneusi and the coccidium in the complex water body of the rural surface sewage channel are synchronously detected, and the difference lies in that the embodiment further includes the following steps:

[0204] The ITS gene site specific primer of the Enterocytozoon bieneusi and the 500bp SSUrRNA gene site specific primer of the coccidium designed by the application are used to detect the total DNA samples of 20 rural surface sewage channels, wherein 6 of the Enterocytozoon bieneusi PCR positive, the bidirectional sequencing results of the genes are all the Enterocytozoon bieneusi, and the known sequences in the GenBank database are compared and analyzed through BLAST (http: / / www.ncbi.nlm.nih.gov), so as to identify the genotypes D (n=3), CHG5 (n=2) and Type IV (n=1) are all known genotypes, and all belong to the zoonotic group Group 1, have the risk of spreading in human, animals and environment, and constitute potential harm to public health; the nucleotide sequences are shown as SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15 respectively.

[0205] The ITS region nucleotide sequence of the Enterocytozoon bieneusi genotype D (n=3) (as shown in SEQ ID NO. 13) is as follows:

[0206] agggatgaag agcttcggct ctgaatatct atggctagat aaagtacaag tcgtaacaag 60 gttcagttg gagaaccagc tgaaggatca ttttcagttt ttggggtgtg ggtatcggaa 120 tgtgtggtag gtgatgtgtg tgtgtatggg ggatgccgag gggaccagcg gtgcggtggt 180 gtgtgcaggc gtgagagtgt atctgcaagt gtgagggatg tgggtgcagc gagttagagg 240 tggttccatg tggaatagtg ggattggtac gtgatggttg gatgggggaa tgatgtgtgt 300 atgggtgagg aaaatcggag gttgcggtgc gagcggcagt agggtgccat caagaggtgt 360 atttggaaat atccctaata caggatcact tggatccgtc ggcg 404

[0207] The ITS region nucleotide sequence of Genotype CHG5 (n=2) of Enterocytozoon bieneusi (as shown in SEQ ID NO. 14) is:

[0208] agggatgaag agcttcggct ctgaatatct atggctagat aaagtacaag tcgtaacaag 60 gttcagttg gagaaccagc tgaaggatca ttttcagttt ttagggtgtg agtatcggaa 120 tgtatagtag gtgatgtatg tgtgtatggg ggatgccgag gggacccgcg gtgcggtggt 180 gtgcggtggt gtgagagtgt atctgtaagt gtgagggatg tagctgcagt gagttagaga 240 gtgttccatg aggaatagtg ggattggtac acgatgggtt gtatggagaa tgatgtgtgt 300 atgggtgagg aaaatcggag gttgcggtgc gagcggcagt agggtgccat caagaggtgt 360 atttggaaat atccctaata caggatcact tggatccgtc ggcg 404

[0209] The ITS region nucleotide sequence of Genotype ype IV (n=l) of Enterocytozoon bieneusi (as set forth in SEQ ID NO. 15) is:

[0210] cttcggctct gaatatctat ggctagataa agtacaagtc gtaacaaggt ttcagttgga 60 gaaccagctg aaggatcatt ttcagttttt ggggtgtggg tatcggaatg tgtggtaggt 120 gatgtgtgtg tgtatggggg atgccgaggg gaccagcggt gcggtggtgt gtgtaggcgt 180 gagagtgtat ctgcaagggt gagggatgtg ggtgcagcga gttagaggtg gttccatgtg 240 gaatagtggg attggtacgt gatggttgga tgggggaatg atgtgtgtat gggtgaggaa 300aatcggaggt tgcggtgcga gcggcagtag ggtgccatca agaggtgtat ttggaaatat 360 ccctaataca ggatcactlg gatccggtcg gc 392

[0211] There were 12 samples positive for Coccidiosis PCR, and the results of bidirectional sequencing of the genes were all Coccidiosis. Subtypes ST10 (n=5), ST3 (n=4), ST7 (n=2) and ST4 (n=1) were identified, and the nucleotide sequences were shown in SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19, respectively.

[0212] The SSUrRNA coding DNA fragment of the identified subtype ST10 (n=5) of Coccidiosis (shown in SEQ ID NO. 16) was as follows:

[0213]

[0214] The ITS region nucleotide sequence of the identified subtype ST3 (n=4) of Coccidiosis (shown in SEQ ID NO. 17) was as follows:

[0215]

[0216] The ITS region nucleotide sequence of the identified subtype ST7 (n=2) of Coccidiosis (shown in SEQ ID NO. 18) was as follows:

[0217]

[0218] The SSU rRNA encoding DNA fragment (as shown in SEQ ID NO. 19) of the identified subtype ST4 (n = 1) of Blastocystis is as follows:

[0219]

[0220] Both Blastocystis and Enterocytozoon bieneusi were identified in 3 water samples. 20 water samples of rural surface sewage ditches were collected from Hainan Province in July-September 2020, each sample was 20L, and a sterile mixed cellulose ester filter membrane with a pore size of 0.22 μm and a diameter of 50 mm was used for vacuum filtration on site. The filter membrane was transported back to the laboratory at low temperature, and the total DNA in the sample was extracted after acetone dissolution as described in the examples of the present application.

[0221] Figure 9 and Figure 10 The gel electrophoresis figures of PCR detection of water samples of rural surface sewage ditches by specific primers of ITS gene site of Enterocytozoon bieneusi and specific primers of 500bp SSUrRNA gene site of Blastocystis of the present application are shown respectively.

[0222] Table 23 (results of bidirectional gene sequencing of PCR amplification products positive for gel electrophoresis) shows the results of bidirectional gene sequencing (Shanghai Biotechnology) and Blast comparison of PCR amplification and gel electrophoresis positive amplification products.

[0223] Table 23

[0224]

[0225]

[0226] In summary, the above-mentioned embodiments of the present application provide a synchronous detection method for detecting Enterocytozoon bieneusi and Blastocystis in complex water bodies. The method focuses on the specific nested PCR amplification of the ITS gene of Enterocytozoon bieneusi and the 500bp SSUrRNA gene of Blastocystis based on specifically designed and optimized primers, agarose gel electrophoresis imaging, bidirectional gene sequencing, and sequence alignment. The presence of Enterocytozoon bieneusi and Blastocystis in the sample is determined based on the electrophoresis results, and the genotypes / new genotypes of Enterocytozoon bieneusi and Blastocystis are identified through gene sequencing. Compared with traditional microscopic observation and other existing detection methods, the above-mentioned method of the present application has higher detection efficiency and sensitivity, can quickly identify the species and genotypes / new genotypes, and has a wide range of applications. The detection results can be further used for genotype / new genotype identification, and the risk of zoonosis and the potential harm to public health can be evaluated based on phylogenetic and genetic evolution analysis. The method can be applied to rapid identification of Enterocytozoon bieneusi and Blastocystis, molecular diagnosis of related intestinal infectious diseases, tracing, prevention and control, and other aspects, and can meet the needs of large-scale, wide-range, low-cost environmental monitoring, public health assessment, and clinical diagnosis.

[0227] It should be noted that in other embodiments of the present application, within the scope of the detection instruments, structures, steps, components, specific primers, ratios, process parameters and conditions, detection objects and the like described in the present application, other different schemes obtained by specific selection can achieve the technical effects described in the present application, therefore the present application does not list them one by one.

[0228] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the scope of the technical solutions of the present application, by using the disclosed methods and technical contents. Any equivalent changes made according to the components, ratios and processes of the present application should be covered by the protection scope of the present application.

Claims

1. A method for simultaneous detection of *Pichia pastoris* and *Blastocystis* in complex water bodies, characterized in that... It includes the following steps: S1: Pretreatment of water samples The method of vacuum filtration, dissolving the filter membrane with acetone, adding PBS buffer, centrifuging and collecting the precipitate was used to enrich microorganisms in complex water samples and remove impurities that may interfere with PCR detection results. S2: Nucleic acid extraction Vortex the precipitate with buffer until fully mixed, add protease, centrifuge and collect the supernatant, and extract whole genomic DNA from the sample using a nucleic acid extraction kit; S3: PCR detection ITS gene site-specific primers were used to amplify *P. pyridostega* by PCR, and 500bp SSUrRNA gene site-specific primers were used to amplify *Blastocystis* by PCR. Positive products were subjected to bidirectional sequencing to qualitatively determine whether *P. pyridostega* and *Blastocystis* were present in the water samples.

2. The method for simultaneous detection of *Pichia pastoris* and *Blastocystis* in complex water bodies according to claim 1, characterized in that, It also includes the following steps: S4: Genotype / Subtype Identification: The positive products of PCR amplification of the two worms were subjected to bidirectional sequencing of their gene sequences. The sequences were then compared with known sequences in the GenBank database using BLAST to further clarify whether they were *Microsporidium pichii* and *Blastocystis*, and to identify the genotype of *Microsporidium pichii* and the subtype of *Blastocystis*.

3. The method for simultaneous detection of *Pichia pastoris* and *Blastocystis* in complex water bodies according to claim 2, characterized in that... It also includes the following steps: S5: Environmental Risk Assessment Phylogenetic and evolutionary analysis of the genotypes / subtypes of *Microsporidium pichii* and *Blastocystis* in the samples was performed using MEGA 11 software. Sequence data were imported, and the Kimura2-paramete model was selected. A phylogenetic tree was constructed using the Neighbor-Joining method, with the Bootstrap Replication count set to 1000 to assess its reliability. The results were exported as images. If the *Microsporidium pichii* genotype identified in the samples belonged to Group 1 (zoonotic) in the phylogenetic tree or had been reported in existing literature for detection in human samples, it indicated that the genotype posed a risk of transmission in humans, animals, and the environment, and a potential threat to public health. If the *Blastocystis* subtype identified in the samples had been reported for detection in human samples, it indicated that the subtype posed a risk of transmission to humans and a potential threat to public health.

4. The method for simultaneous detection of *Pichia pastoris* and *Blastocystis* in complex water bodies according to claim 1, characterized in that, In step S1, the filter membrane used for filtration is a sterile mixed cellulose ester filter membrane with a diameter of 50 mm and a pore size of 0.22 μm, in order to enrich microorganisms in complex water samples and intercept all microorganisms in the water except for viruses, and remove interfering impurities; acetone is an analytical grade reagent.

5. The method for simultaneous detection of *Pichia pastoris* and *Blastocystis* in complex water bodies according to claim 1, characterized in that... In step S2, whole-genome DNA is extracted from the sample using the QIAamp Fast DNA Stool Mini kit, specifically including the following steps: S2-1: Transfer the thawed precipitate sample into a 2mL centrifuge tube, add 1mL of [missing information]. Buffer, vortex until thoroughly mixed. Then, heat the sample in a 95°C metal bath for 5 min, vortex for 15 s, and centrifuge at 14000 r / min for 1 min at 4°C; S2-2: Take a 1.5 mL sterile centrifuge tube and add 15 μL proteinase K, 200 μL of centrifuged sample supernatant, and 200 μL of AL buffer in sequence. Vortex for 15 seconds and then place the mixed liquid sample in a 70℃ metal bath for 10 min. S2-3: Add 200 μL of anhydrous ethanol, vortex to mix, then add the mixture to the filter column and place the filter column into a new 2 mL collection tube. Mark the filter column cap and centrifuge at 14000 r / min for 1 min at 4 °C. S2-4: Remove the filter column after centrifugation, transfer the filter column to a new 2mL collection tube, add 500μL of AW1 buffer, and centrifuge a second time at 14000r / min for 1min at 4℃. S2-5: Remove the filter column after centrifugation again, put it into a new 2mL collection tube, add 500μL of AW2 buffer, and centrifuge for the third time at 14000r / min for 1min at 4℃. S2-6: Transfer the centrifuged filter column to a new 2mL collection tube and centrifuge for the fourth time at 14000r / min for 3min at 4℃; S2-7: Finally, remove the filter column, transfer it to a new 1.5mL centrifuge tube, add 200μL of ATE buffer directly, incubate at room temperature for 1min, centrifuge at 14000r / min for 2min at 4℃, elute to obtain DNA, and store at -80℃ for later use.

6. The method for simultaneous detection of *Pichia pastoris* and *Blastocystis* in complex water bodies according to claim 1, characterized in that... In step S3, the PCR detection uses specific primers to amplify *P. praecox* and *Blastocystis* respectively. The positive products are bidirectionally sequenced to qualitatively determine whether *P. praecox* and *Blastocystis* are present in the water sample. *P. praecox* positive samples and *Blastocystis* positive samples identified by bidirectional gene sequencing are used as positive controls, and DEPC water is used as a negative control. The PCR reaction system comprises: 2 μL of whole genomic DNA as template, 0.5 μL each of forward and reverse primers for specific amplification, 0.5 μL of 5 U / μL Taq DNA polymerase, 2.5 μL of 10× Buffer, 2 μL of 10 mM dNTPs, and 17 μL of DEPC water, for a total volume of 25 μL; specifically including the following steps: S3-1: PCR detection of the ITS gene locus in *Microsporidium pichii* The specific primers for the first round of PCR amplification of *Microsporidium pichii* are as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; The first round of PCR amplification reaction program was as follows: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 40 s, for a total of 35 cycles; and finally 72℃ extension for 10 min. The specific primers for the second round of PCR amplification of *Microsporidium pichii* are as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4; The second round of PCR amplification reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, for a total of 35 cycles; and finally 72℃ extension for 10 min. S3-2: PCR detection of the 500bp SSUrRNA gene locus in *Blastocystis* The specific primers for the 500bp SSUrRNA gene locus are as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.9, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.10; The PCR amplification program for Blastomyces was as follows: denaturation at 95℃ for 4 min; denaturation at 95℃ for 30 s, annealing at 54℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles; and a final extension at 72℃ for 5 min.

7. The method for simultaneous detection of *Pichia pastoris* and *Blastocystis* in complex water bodies according to claim 1, characterized in that, Step S4 specifically includes the following steps: S4-1: Determine whether it is the genotype of the two parasites: The positive products of PCR amplification of the two parasites were subjected to bidirectional sequencing of gene sequences. The sequences were compared with known sequences in the GenBank database by BLAST to further clarify whether they are the genotypes of Microsporidium pichii and Protozoa blastosa. S4-2: Identification of new genotypes of *Microsporidium pichii*: After confirming the genotype of *Microsporidium pichii*, according to the genotyping rules of *Microsporidium pichii*, if one base in the 243-nucleotide sequence of the *Microsporidium pichii* ITS region detected in the sample is different from any known sequence in the GenBank database, it is identified as a new genotype of *Microsporidium pichii*. S4-3: Identification of new subtypes of *Blastocystis*: After confirming the genotype of *Blastocystis*, according to the identification criteria for new subtypes of *Blastocystis*, the new subtype is identified by comparing ≥80% of the CA bases in the 1800bp SSU nucleotide sequence. If the new subtype differs from all known sequences in the GenBank database by ≥4% and is not on the same branch as any known subtype on the phylogenetic tree, it is identified as a new subtype of *Blastocystis*.

8. The method for simultaneous detection of *Pichia pastoris* and *Blastocystis* in complex water bodies according to claim 1, characterized in that, The environmental risk assessment in step S5 specifically includes the following steps: S5-1: Constructing a phylogenetic tree using MEGA 11 software: Import sequence data, select Kimura 2-paramete as the parametric model, and use the Neighbor-Joining method to construct the tree. Set the BootstrapReplications to 1000 to evaluate the reliability of the tree, and export the results as an image. S5-2: Search and compare with the NCBI database. If the Enterospora bifidum and Bacillus spp. identified in the sample have been reported to be detected in human samples, it indicates that the subtype has the risk of transmission to humans and poses a potential threat to public health.

9. The application of the method for simultaneous detection of *Pichia pastoris* and *Blastocystis* in complex water bodies according to any one of claims 1 to 8, characterized in that, Based on the phylogenetic and genetic evolutionary analysis of *Microsporidium pichii* and *Blastocystis*, if the *Microsporidium pichii* genotype identified in the sample belongs to Group 1 (zoonotic group) in the phylogenetic tree or has been reported in existing literature to have been detected in human samples, it indicates that the genotype has the risk of transmission in humans, animals, and the environment and poses a potential threat to public health. If the *Blastocystis* subtype identified in the sample has been reported to have been detected in human samples, it indicates that the subtype has the risk of transmission to humans and will pose a potential threat to public health.

10. The application according to claim 9, characterized in that, This simultaneous detection method was applied to the rapid identification of *Pichia pastoris* and *Blastocystis*, the molecular diagnosis, source tracing, and prevention of related intestinal infectious diseases.

Images