Rabbit encephalitis microsporidia nested PCR (polymerase chain reaction) specific primer as well as detection method and application thereof

Abstract

The invention discloses a rabbit encephalitis microsporidia nested PCR specific primer as well as a detection method and application thereof, and relates to the technical field of rabbit encephalitis microsporidia detection. The invention relates to a nested PCR (Polymerase Chain Reaction) specific primer for rabbit encephalitis microsporidia. A gene sequence of an outer forward outer primer is shown as SEQ ID NO.2; the gene sequence of the outer reverse outer primer is as shown in SEQ ID NO.3; the gene sequence of the inner side forward inner primer is as shown in SEQ ID NO. 4; and the gene sequence of the inner side forward outer and inner primers is as shown in SEQ ID NO. 5. The nested PCR primer designed by the invention is high in specificity, can only identify E.cloniculi, has no cross reaction with related species, has sensitivity improved by 100 times compared with that of conventional PCR, can detect more low-concentration positive samples, and reduces the omission ratio; the matched kit fills the market blank, and provides efficient and accurate technical support for clinic and prevention and control.

Description

Technical Field

[0001] This invention relates to the field of rabbit encephalitis microsporidia detection technology, specifically to a rabbit encephalitis microsporidia nested PCR specific primer, its detection method, and its application. Background Technology

[0002] Rabbit encephalitis microsporidia ( E.cuniculi As an obligate intracellular opportunistic pathogen with a wide host range, it can be transmitted through environmental media such as contaminated water sources, soil, and fresh food, posing a threat to a variety of mammals, including humans, as well as birds and fish. Its infection can cause severe encephalitis microsporidiosis and even death. Therefore, efficient detection of it is of great significance.

[0003] Currently, the detection methods for rabbit encephalitis microsporidia mainly rely on staining microscopy (such as Gram staining, Giemsa staining, and modified trichrome staining), serological methods (such as IFA, ELISA, and Western blot), conventional PCR, and nested PCR technology. However, these detection methods all have certain limitations, which seriously restrict the accurate identification and efficient control of this pathogen. Specifically: (1) The staining microscopy method is cumbersome, time-consuming and dependent on the operator's experience. At the same time, microsporidia spores are similar in morphology to other non-pathogenic fungi, yeasts, bacteria and even some impurities under an optical microscope, making it difficult to distinguish them and making species identification impossible, which may lead to missed detection or false positives.

[0004] (2) Serological methods have problems such as low sensitivity, complicated operation and possible significant cross-reactivity, which make it difficult to meet the needs of accurate detection.

[0005] (3) Although conventional PCR is relatively simple to operate, its sensitivity is still insufficient when detecting trace samples, and it cannot effectively cope with the detection scenario of low concentration of pathogens.

[0006] (4) Although nested PCR technology can significantly improve detection sensitivity and specificity compared to conventional PCR, there are currently no commercially available nested PCR technologies. E.cuniculi Nested PCR detection kits limit the widespread application of this technology.

[0007] (5) In addition, some reported nested PCR primers are mainly for the genus Microsporidia ( Encephalitozoon ),include E.cuniculi and its closely related species (such as E. hellem、E. intestinalis The universal primers designed are difficult to reliably distinguish individually. E. cuniculi, Non-specific amplification products are easily generated during the amplification process, affecting the specificity and accuracy of detection.

[0008] Therefore, the inventors believe that how to solve the above-mentioned technical problems is a key issue that urgently needs to be addressed by those skilled in the art.

[0009] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides a nested PCR specific primer for rabbit encephalitis microsporidia pathogens, along with its detection method and application, to resolve the issues raised in the background section.

[0011] A specific primer for nested PCR of rabbit encephalitis microsporidia. The gene sequence of the outer forward primer is shown in SEQ ID NO.2; SEQ ID NO.2 is 5'-GGAGGGGATGGAAGAGAACAGGTC-3'; The gene sequence of the outer reverse primer is shown in SEQ ID NO.3; SEQ ID NO.3 is 5'-ATGCCATTTCACTCGCCGCTACTC-3'; The gene sequence of the inner forward primer is shown in SEQ ID NO.4; SEQ ID NO.4 is 5'-TTGAATGTGTCCCCTGTCCTTTGTA-3'; The gene sequence of the inner reverse primer is shown in SEQ ID NO.5; SEQ ID NO.5 is 5'-ACAAACAAACAAACATCCATCAAA-3'.

[0012] The characteristics of the above primer sets are shown in Table 1.

[0013] Table 1. Characteristics of nested PCR primers (the positions are based on sequence SEQ ID NO: 1).

[0014]

[0015] A method for detecting rabbit encephalitis microsporidia nested PCR specific primers as described above includes the following steps: Genomic DNA was extracted from the sample to be tested; The extracted genomic DNA template was amplified using the outer primer pair in the first round of PCR. The product from the first round of PCR amplification was diluted 10-100 times and used as a template for the second round of PCR amplification using the inner primers. Finally, the second round of PCR amplification products were detected by agarose gel electrophoresis to analyze whether the target band of 183bp was present.

[0016] Preferably, the annealing temperature of the outer primer during PCR amplification is 60°C; and the annealing temperature of the inner primer during PCR amplification is 55°C.

[0017] Preferably, the concentrations of both the outer and inner primers in their respective systems are 0.2 μM.

[0018] Application of a nested PCR-specific primer for rabbit encephalitis microsporidia as described above in the preparation of products for detecting rabbit encephalitis microsporidia.

[0019] Application of a rabbit encephalitis microsporidia nested PCR specific primer as described above in the preparation of a rabbit encephalitis microsporidia detection kit.

[0020] A kit for detecting rabbit encephalitis microsporidia, the kit comprising the rabbit encephalitis microsporidia nested PCR specific primers described above.

[0021] Preferably, it also includes PCR reaction reagents; the PCR reaction reagents contain DNA polymerase, dNTPs, Mg²⁺, buffer, etc.

[0022] The present invention provides a nested PCR specific primer for rabbit encephalitis microsporidia, its detection method, and its application, which have the following beneficial effects: The nested PCR primers designed in this invention have high specificity and can only identify... E.cuniculi It exhibits no cross-reactivity with closely related species, and its sensitivity is 100 times higher than that of conventional PCR, with a detection limit of 0.01237 pg / μL (12.37 fg / μL). It can detect more low-concentration positive samples and reduce the false negative rate. The accompanying kit fills a market gap and provides efficient and accurate technical support for clinical and prevention and control. Attached Figure Description

[0023] Figure 1 Electrophoresis diagram showing the optimized annealing temperature for the nested PCR reaction of this invention; wherein, Figure 1 Image A shows the electrophoresis result of the first round of PCR annealing temperature gradient. Figure 1 Image B in the image is an electrophoresis diagram of the second round of PCR annealing temperature gradient.

[0024] Figure 2 This is an electrophoresis image used to verify the specificity of the nested PCR primer set of this invention.

[0025] Lane descriptions: M: DNA molecular weight standard (100-3000 bp); P: Positive control; N: Negative control; 1: E. cuniculi DNA template; 2: E. hellem DNA template; 3: E. intestinalis DNA template; 4: E. bieneusi DNA template; 5: E. coli DNA template.

[0026] Figure 3 This is an electrophoresis diagram comparing the sensitivity of nested PCR and conventional PCR in this invention. Figure 3 A represents the result of nested PCR electrophoresis; Figure 3 B represents the result of routine PCR electrophoresis.

[0027] Lane descriptions: M: DNA molecular weight standard (100-3000 bp); P: Positive control; N: Negative control; 3-10: E. cuniculi genomic DNA template, with final concentrations of 123.7 pg / μL, 12.37 pg / μL, 1.237 pg / μL, 0.1237 pg / μL, 0.01237 pg / μL, 0.001237 pg / μL, and 0.0001237 pg / μL, respectively.

[0028] Figure 4 This is an electrophoresis diagram showing the detection results of clinical samples using the nested PCR method of this invention.

[0029] Lane description: M: DNA molecular weight standard (100-3000 bp); P: Positive control; N: Negative control; 1-15: Detection results of sample numbers 1-15 (of which samples 3, 6, 8, 9, 12, 14, and 15 are positive).

[0030] Figure 5 Electrophoresis diagram of the detection results of clinical samples as a comparative example (conventional PCR method).

[0031] Lane description: M: DNA molecular weight standard (100-3000 bp); P: Positive control; N: Negative control; 1-15: Detection results of samples 1-15 (samples 3, 8, 9, and 14 are positive). Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] I. The design concept of the specific primers for nested PCR of rabbit encephalitis microsporidia, its detection method, and its application provided in the embodiments of the present invention is as follows: (1) Precise selection of target region: SEQ ID NO.1 (rDNA gene cluster fragment) was selected as the target for the following reasons: rDNA exists in the genome in multiple copies, typically 100-1000 copies, far exceeding single-copy protein genes, which can significantly improve PCR amplification efficiency; multiple copies of the template are more easily bound by primers, making them suitable for the detection of small samples.

[0034] SEQ ID NO.1 contains: a portion of SSUrRNA (conserved region) + the complete ITS1 (hypervariate region) + a portion of LSUrRNA (conserved region); SSUrRNA and LSUrRNA are highly conserved sequences within the species, and their positions differ. E.cuniculi The differences between strains are minimal, ensuring that the primers can stably bind to all strains.

[0035] Meanwhile, ITS1 (interspecific spacer region) is a highly variable sequence across species, and its position ensures... E.cuniculi and closely related species E. hellem、E. intestinalis The ITS1 of these species differs significantly and serves as a molecular barcode for distinguishing species.

[0036] Therefore, the selection of target regions itself embodies the design logic of "conservatism ensures universality, and high-variability regions ensure specificity," laying the foundation for primer screening.

[0037] (2) When screening outer primers, the following conditions must be met: the amplification range (2730-3264bp) of the outer primer pair must completely contain the complete ITS1 region (3049-3083bp) and some conserved SSU / LSUrRNA to ensure that the primary amplification product contains both species-specific sequence (ITS1) and has sufficient length for secondary amplification by the inner primers.

[0038] By comparing 10 plants in the NCBI database E.cuniculi The rDNA sequences (such as AJ005581.1, KF169728, etc.) were used to confirm that the outer primer binding regions (2730-2753bp and 3241-3264bp) were completely conserved in all strains and had no base variations, thus avoiding missed detections due to strain differences.

[0039] And, the outer primer sequence is compared with closely related species ( E. hellem、E. intestinalis The rDNA sequences of (e.g., 3250-3270 bp) were compared using BLAST to ensure that the primers differed from those of other species by more than three bases, such as the 3250-3270 bp region of the outer reverse primer. E.hellem The homologous region has 5 base mismatches, which reduces the risk of nonspecific binding from the source.

[0040] (3) When screening inner primer pairs, the following conditions must be met: the binding regions of the inner primers (2895-2918bp and 3060-3083bp) must be completely located within the amplification range of the outer primers (2730-3264bp), and they must be separated from the outer primers by 50-100bp to avoid interference between primers. This design ensures that even if the outer primers have a small amount of non-specific amplification (such as binding to host DNA), the inner primers will not be able to amplify because they have no binding sites, thus achieving dual specificity screening.

[0041] The binding region of the inner reverse primer (3060-3083bp) directly covers the 3' end of ITS1 (3049-3083bp), and ITS1 is the region with the most significant interspecific differences. E.cuniculi and E.intestinal (The ITS1 sequence similarity is only 65%); by anchoring the inner primer to the ITS1 region, the specificity for the target species is further improved.

[0042] The inner primer sequences were adjusted by GC content (45%-55%) and secondary structure prediction (no hairpin structures or primer dimers) to ensure amplification efficiency of over 90% (common primer efficiency is usually 70%-80%). Combined with a 10-fold diluted primary product template, the signal was amplified 100 times (sensitivity increased from 1.237 pg / μL in conventional PCR to 0.01237 pg / μL).

[0043] The inner and outer primers were synergistically optimized through screening experimental conditions to balance amplification efficiency and specificity, as detailed below: Four annealing temperature gradients were designed for the first round of amplification (45-60℃) and the second round of amplification (50-65℃). The brightness of the amplified bands and non-specific products were detected by electrophoresis. Finally, the outer primer was selected at 60℃ and the inner primer at 55℃. The annealing temperature of the inner primer was 5℃ lower to avoid competition for template with the outer primer.

[0044] After testing multiple primer concentrations, it was found that the amplified band was clearest at 0.2 μM, so the final concentration was determined to be 0.2 μM.

[0045] A total of 8 pairs of candidate primers (4 pairs of outer primers and 4 pairs of inner primers) were synthesized, and specificity experiments were conducted to detect... E. hellem、 E. intestinalis、 E. bieneusi These non-target microorganisms were compared, and other primers that had cross-reactivity with non-target microorganisms were eliminated. Finally, the nested PCR primers composed of SEQ ID NO. 2-5 were determined as the best primers.

[0046] II. To address the aforementioned technical problems, this invention provides a specific primer for nested PCR of rabbit encephalitis microsporidia, along with its detection method and application, to solve the problems mentioned in the background section. Specific embodiments are as follows: Example 1: Extraction of rabbit encephalitis microsporidia from samples using the CTAB method E.cuniculi DNA Add 300 mg of fecal sample to a 2 ml centrifuge tube, add 1.5 ml of 2% CTAB solution + 150 μL of proteinase K to each tube and vortex thoroughly to mix. Incubate at 56 °C for at least 4 hours. Centrifuge at 12000 rpm for 6 min, transfer the supernatant to a new 2 ml test tube and add 700 μL of DNA extraction buffer (Tris-saturated phenol, chloroform and isoamyl alcohol mixed in a 25:24:1 ratio), vortex for 3 min, and centrifuge at 13000 rpm at 4℃ for 10 min. (3) Transfer the supernatant to a new 1.5ml test tube and add 1ml of isopropanol, then allow it to settle at -20℃ for at least 1 hour; (4) Centrifuge at 13000 rpm and 4℃ for 10 min, then discard the supernatant; (5) Wash with 600 μL of 75% ethanol, centrifuge at 13000 rpm and 4℃ for 10 min, discard the supernatant, and repeat once; (6) Open the test tube cap and allow the ethanol to evaporate under natural conditions; (7) Add 50 μL of ddH2O to each tube and measure the concentration and purity.

[0047] Example 2: Optimization of the optimal annealing temperature for nested PCR primers (inner and outer primers) The first round of PCR reaction system is shown in Table 2; the reaction procedure is shown in Table 3, where the annealing temperature is designed with four temperature gradients of 50℃, 55℃, 60℃ and 65℃. Table 2. Nested PCR first-round reaction system

[0048] Table 3. Nested PCR First Round Reaction Procedure

[0049] Note: X represents four annealing temperature gradients: 50, 55, 60, and 65.

[0050] Second round PCR reaction: The first round PCR product was diluted 10-fold with sterile ddH2O and used as a template. The reaction system is shown in Table 4 below. The reaction program is shown in Table 5 below, where the annealing temperature was set at four temperature gradients: 45℃, 50℃, 55℃, and 60℃.

[0051] Table 4. Nested PCR second-round reaction system

[0052] Table 5. Nested PCR Second Round Reaction Procedure

[0053] Note: Y represents the four annealing temperature gradients of 45, 50, 55, and 60.

[0054] The amplification products of the first and second PCR were detected by agarose gel electrophoresis, respectively. (1) Prepare buffer solution for 0.5×TBE electrophoresis and gel preparation; (2) Add 1.7g of agarose to 100ml of 0.5×TBE buffer and heat to dissolve the agarose completely and evenly; (3) Pour the agarose solution into the gel mold, and then insert the comb at the appropriate position. The gel thickness is between 3 and 5 mm. (4) Allow the gel to solidify at room temperature for 30 to 40 minutes, then remove the comb and spot the sample. (5) Mix 5 μL of PCR amplification product with 1 μL of 6× DNA Loading Buffer thoroughly, and then spot the samples sequentially. The amount of DNA Marker (100~3000 bp) spotted is 8 μL. (6) Place in an electrophoresis tank and perform electrophoresis at 100V for 40-60 minutes; (7) The results were photographed and analyzed using a gel imaging system. Samples with amplification products of the first and second PCR at 532bp and 183bp were initially screened as positive.

[0055] 4. Experimental Results: By comparing the amplification efficiency and specificity at different annealing temperatures (see results below) Figure 1 Finally, the optimal annealing temperature for the first round of PCR was determined to be 60℃, and the optimal annealing temperature for the second round of PCR was determined to be 55℃.

[0056] Example 3: Verification of the specificity of inner and outer primers in nested PCR 1. Extract separately E. cuniculi (Positive control) E. hellem、E. intestinalis、E. bieneusi、E. coli And genomic DNA from sterile ddH2O (negative control).

[0057] 2. Using the eight candidate nested PCR primer pairs (four outer primer pairs and four inner primer pairs) obtained from the initial screening, the template was subjected to the first round of PCR amplification. The reaction system is shown in Table 2 of Example 2, and the reaction procedure is shown in Table 3 of Example 2.

[0058] 3. The reaction system for the second round of PCR is shown in Table 4, and the reaction procedure is shown in Table 5. The first amplification product diluted 10-fold is used as template DNA. 4. The amplification products of the second PCR were detected by agarose gel electrophoresis. Specificity results showed that the nested PCR primers consisting of SEQ ID NO. 2-5, namely SEQ ID NO. 2 (outer forward primer), SEQ ID NO. 3 (outer reverse primer), SEQ ID NO. 4 (inner forward primer), and SEQ ID NO. 5 (inner reverse primer), were only effective for [specific PCR products]. E.cuniculi A specific band of 183 bp was amplified in the sample, while the negative control and closely related species ( E. hellem、E. intestinalis、 E.bieneusi When the samples were tested, no amplification bands were observed, indicating that the primer pair has strict species specificity. Figure 2 The detection results of the nested PC primer pairs consisting of SEQ ID NO. 2-5 are shown.

[0059] However, other candidate primer pairs all exhibited significant non-specific amplification issues during detection: they could amplify not only... E.cuniculi The target band may also interact with closely related non-target microorganisms (such as...) E. hellem、E. intestinalis、 E.bieneusi Cross-reactions can occur, amplifying non-specific bands and leading to distorted detection results.

[0060] In contrast, nested PCR primer pairs consisting of SEQ ID NO. 2-5 specifically recognize only... E.cuniculi The primer pair amplified a 183 bp target band, which showed no cross-reactivity with the aforementioned non-target microorganisms, resulting in accurate and reliable detection. Therefore, this primer pair was determined to be the optimal choice.

[0061] Example 4: Validation of Nested PCR Sensitivity 1. The reaction system for the first round of nested PCR is shown in Table 2; the reaction procedure is shown in Table 3 (annealing temperature 60℃). 2. The amplification template for the first round of nested PCR was DNA serially diluted 10-fold, with final concentrations of 123.7 pg / μL, 12.37 pg / μL, 1.237 pg / μL, 0.1237 pg / μL, 0.01237 pg / μL, 0.001237 pg / μL, and 0.0001237 pg / μL. 3. The nested PCR second reaction system is shown in Table 4, and the reaction procedure is shown in Table 5 (annealing temperature 55℃). The first amplification product is used as the template DNA. The conventional PCR amplification system is shown in Table 6 below, and the reaction procedure is the same as in Table 3 of Example 2 (annealing temperature 60℃), but the number of cycles is increased to 35. The DNA template is the same as the DNA template used in the first nested PCR reaction. Table 6 Conventional PCR Reaction System

[0062] 4. Agarose gel electrophoresis was used to detect the products of the second reaction of nested PCR and conventional PCR amplification, respectively. Conventional PCR only detected a clear expected band (532 bp) when the template concentration was higher than or equal to 1.237 pg / μL. See the results below. Figure 3 Lane B, lane 3. Nested PCR still produced clear, specific bands even at template concentrations as low as 0.01237 pg / μL; see results below. Figure 3 Lane A, Lane 5. Calculations show that the detection sensitivity of the nested PCR method established in this invention is 100 times higher than that of the conventional PCR method.

[0063] 5. Example 5: Comparison of detection results of nested PCR and conventional PCR samples To verify the effectiveness of the method of the present invention in actual sample detection, the following experiment was conducted: 1. Sample and DNA extraction: Fifteen fecal samples from pet rabbits were collected, and genomic DNA was extracted using the CTAB method described in Example 1.

[0064] 2. Nested PCR amplification: First round of PCR: The reaction system was the same as in Table 2 of Example 2, and the reaction procedure was the same as in Table 3 of Example 2 (annealing temperature 60℃). 15 DNA samples to be tested, one known E. cuniculi positive DNA sample (positive control), and one ddH2O sample (negative control) were used as templates.

[0065] Second round PCR: The product from the first round PCR was diluted 10-fold with ddH2O and used as a template. The reaction system was the same as in Table 4 of Example 2, and the reaction procedure was the same as in Table 5 of Example 2 (annealing temperature 55℃).

[0066] 3. Conventional PCR amplification (comparison method): The reaction system was the same as in Table 6 of Example 4, and the reaction procedure was the same as in Table 3 of Example 2 (annealing temperature 60°C), but the number of cycles was increased to 35 cycles, and the template was the same as the template used in the first round of nested PCR.

[0067] 4. Electrophoresis detection: Take 5 μL of the product from the second round of nested PCR and the product from conventional PCR, respectively, and perform agarose gel electrophoresis analysis.

[0068] 5. Results Analysis: The positive control showed the expected band, while the negative control showed no band, indicating that the experimental results are reliable. The test results of 15 samples showed that nested PCR detected 7 positive samples (samples 3, 6, 8, 9, 12, 14, and 15). See [link to relevant documentation]. Figure 4 Three positive samples (samples 3, 8, and 14) were detected by conventional PCR. These results indicate that the nested PCR detection method and primer combination (SEQ ID NO: 2-5) established in this invention can be used for the detection of actual E. cuniculi samples, and its detection sensitivity is higher than that of conventional PCR methods.

[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A specific primer for nested PCR of rabbit encephalitis microsporidia, characterized in that, The gene sequence of the outer forward primer is shown in SEQ ID NO.2; The gene sequence of the outer reverse primer is shown in SEQ ID NO.3; The gene sequence of the inner forward primer is shown in SEQ ID NO.4; The gene sequence of the inner reverse primer is shown in SEQ ID NO.

5.

2. A method for detecting rabbit encephalitis microsporidia nested PCR specific primers as described in claim 1, characterized in that, Includes the following steps: Genomic DNA was extracted from the sample to be tested; The extracted genomic DNA template was amplified using the outer primer pair in the first round of PCR. The product from the first round of PCR amplification was diluted and used as a template for the second round of PCR amplification using the inner primers. Finally, the second round of PCR amplification products were detected by agarose gel electrophoresis.

3. The method for detecting rabbit encephalitis microsporidia nested PCR specific primers according to claim 2, characterized in that, The annealing temperature of the outer primer during PCR amplification is 60℃; the annealing temperature of the inner primer during PCR amplification is 55℃.

4. The method for detecting rabbit encephalitis microsporidia nested PCR specific primers according to claim 2, characterized in that, The concentrations of both the outer and inner primers in their respective systems were 0.2 μM.

5. The application of the rabbit encephalitis microsporidia nested PCR specific primer as described in claim 1 in the preparation of products for detecting rabbit encephalitis microsporidia.

6. The application of the rabbit encephalitis microsporidia nested PCR specific primer as described in claim 1 in the preparation of a rabbit encephalitis microsporidia detection kit.

7. A kit for detecting rabbit encephalitis microsporidia, characterized in that, The kit includes the rabbit encephalitis microsporidia nested PCR specific primers as described in claim 1.

8. The kit for detecting rabbit encephalitis microsporidia according to claim 7, characterized in that, It also includes PCR reaction reagents.

Images