Primer combinations and applications for isothermal amplification-LFD rapid detection of porcine epidemic diarrhea virus G2 gene subtype.

Abstract

This invention discloses a primer combination and its application for isothermal amplification-LFD rapid detection of porcine epidemic diarrhea virus (PEDV) G2 gene subtype, belonging to the field of animal viral molecular biology testing methods. This invention designs a primer combination targeting the S gene of PEDV. By setting a mismatched base at the 3' end characteristic base site of the upstream primer in this primer combination, the amplification specificity of the PEDV G2c gene subtype target sequence is significantly improved. Based on this primer combination, a rapid detection kit for isothermal amplification-LFD of PEDV G2c gene subtype was designed. This kit enables rapid identification and detection of PEDV G2c gene subtype strains, as well as specific and sensitive detection of the PEDV G2c gene subtype, which is of great significance for the diagnosis and epidemiological investigation of PEDV.

Description

Technical Field

[0001] This invention relates to the field of molecular biological testing methods for animal viruses, and in particular to primer combinations and applications for rapid detection of porcine epidemic diarrhea virus G2 gene subtype by isothermal amplification-LFD. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV), which is particularly harmful to suckling piglets, with mortality rates often reaching 80%-100%. It is one of the major animal diseases in the pig industry that is difficult to control and has the highest number of reported cases. As a member of the coronavirus family, PEDV has a single-stranded positive-sense RNA genome that mutates frequently. Based on the S gene sequence, it is currently mainly divided into several genotypes and subtypes, such as G1 and G2, among which the G2c genotype has become the dominant circulating strain in China since 2020. Due to the weak cross-protection between different genotypes, and the fact that conventional genotyping relies on sequencing and evolutionary analysis, which is cumbersome and takes more than 3 days, there is an urgent need to establish a detection method suitable for rapid field identification of the G2c genotype. This is of great significance for precise epidemic control and vaccine compatibility assessment.

[0003] Mutant amplification arrest PCR is based on the fact that Taq DNA polymerase cannot repair a single base mismatch at the 3' end of the primer, thus blocking amplification and enabling the detection of mutant genotypes. This method can be applied to SNP detection, but considering that a single base mismatch can still lead to inefficient extension amplification, in practical applications, other mismatched bases are usually introduced near the 3' end base to complement the 3' end SNP base, improving the specificity of primer amplification. Recombinase-mediated isothermal amplification (MIRA) utilizes recombinase to target the primer to the target sequence. Similar to mutant amplification arrest PCR, it can target the target sequence by creating a base mismatch near the 3' end SNP base. In addition, MIRA combined with lateral flow chromatography (LFD) strips is a nucleic acid on-site detection technology developed in recent years. Compared with traditional real-time quantitative PCR, this technology does not require a thermal cycler and can achieve efficient amplification of the target sequence under isothermal conditions. The amplification products can be quickly visualized and interpreted through LFD. The overall operation is simple and the detection time is short, making it particularly suitable for rapid diagnosis at the grassroots level and in the field. MIRA technology utilizes recombinases and primers to form protein-nucleic acid complexes, which scan DNA templates at room temperature and initiate strand displacement amplification. With specific primer modifications (such as FAM and biotin labeling), the products can be captured and visualized by LFD, exhibiting high sensitivity and specificity. Currently, there are no patents or literature reports on rapid on-site genotyping of PEDV G2c genotypes based on MIRA-LFD technology. Summary of the Invention

[0004] The purpose of this invention is to provide primer combinations and applications for isothermal amplification-LFD rapid detection of porcine epidemic diarrhea virus (PEDV) G2 gene subtype, thereby addressing the problems existing in the prior art. By designing specific primer combinations targeting the S gene, and based on these primer combinations, an isothermal amplification-LFD rapid detection kit was designed. This kit can be used to rapidly identify PEDV G2c gene subtype strains, playing an important role in the rapid on-site detection and prevention of PEDV.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a primer combination for rapid detection of porcine epidemic diarrhea virus G2c gene subtype isothermal amplification-LFD, wherein the primer combination consists of an upstream primer with nucleotide sequences as shown in SEQ ID NO.1 and a downstream primer with SEQ ID NO.2.

[0006] Preferably, the 5' end of the upstream primer is labeled with a 6-carboxyfluorescein group, and the 5' end of the downstream primer is labeled with a biotin group.

[0007] The present invention also provides a rapid detection kit for isothermal amplification-LFD of porcine epidemic diarrhea virus G2c gene subtype, comprising the primer combination described above.

[0008] Preferably, the kit further includes: lyophilized powder, amplification buffer, sterile deionized water, lateral flow chromatography test strips, negative control, and positive control.

[0009] The present invention also provides the application of the primer combination described herein in the preparation of products for detecting the G2c gene subtype of porcine epidemic diarrhea virus.

[0010] Preferably, the detection is performed as follows: using the genomic RNA of the porcine epidemic diarrhea virus sample to be tested as a template, isothermal amplification is performed using the upstream primer and the downstream primer, the amplification product is detected using a lateral flow chromatography test strip, and the detection result of the lateral flow chromatography test strip is used to determine whether the porcine epidemic diarrhea virus sample to be tested contains the porcine epidemic diarrhea virus G2c gene subtype.

[0011] Preferably, the method of judgment is as follows: when the detection line of the lateral flow chromatography test strip turns red, it is judged as positive, that is, the porcine epidemic diarrhea virus sample to be tested contains the porcine epidemic diarrhea virus G2c gene subtype. When the test line of the lateral flow chromatography test strip shows no color development, it is judged as negative, that is, the porcine epidemic diarrhea virus sample to be tested does not contain the porcine epidemic diarrhea virus G2c gene subtype.

[0012] The present invention also provides the application of the primer combination described herein in the preparation of products for diagnosing infection with the G2c gene subtype of porcine epidemic diarrhea virus.

[0013] Preferably, the diagnosis is as follows: using the genomic RNA of the sample to be tested as a template, isothermal amplification is performed using the upstream primer and the downstream primer, the amplification product is detected using a lateral flow chromatography test strip, and the test result is used to determine whether the sample to be tested is infected with the porcine epidemic diarrhea virus G2c gene subtype.

[0014] Preferably, the method of judgment is as follows: when the detection line of the lateral flow chromatography test strip turns red, it is judged as positive, that is, the sample to be tested is a sample infected with the G2c gene subtype of porcine epidemic diarrhea virus; When the test line of the lateral flow chromatography test strip shows no color development, it is judged as negative, that is, the sample to be tested is a sample that has not been infected by the porcine epidemic diarrhea virus G2c gene subtype.

[0015] The present invention discloses the following technical effects: This invention compares and analyzes the S gene sequences of different genotypes of PEDV, selects the characteristic base sites that distinguish the G2c genotype from other genotypes, and designs them to be placed at the 3' end of the upstream primer of G2c, so that the primer specifically recognizes the G2c genotype sequence.

[0016] This invention utilizes isothermal amplification technology to rapidly amplify the S gene of porcine epidemic diarrhea virus (PEDV) based on RNA nucleic acid materials. The detection results are presented using lateral flow chromatography test strips, reducing reliance on large instruments and meeting the needs of on-site real-time testing. Based on the specific recognition capability of the primer pairs in this invention, strains of the G2c gene subtype of PEDV can be specifically detected, shortening the time for traditional strain isolation and identification and reducing detection costs. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A comparison diagram of primer designs for the isothermal amplification-LFD rapid detection kit for porcine epidemic diarrhea virus G2c subtype; the black boxes mark the G2c-F and G2c-R primer pairs, and the base sites specific to the G2c strain are also marked. Figure 2Figure 1 shows the results of the reaction temperature optimization experiment for porcine epidemic diarrhea virus G2c subtype MIRA-LFD; the reaction temperatures corresponding to the six test strips from left to right are 36℃, 38℃, 40℃, 42℃, 44℃, and 46℃, respectively; N is the negative control. Figure 3 The figure shows the results of the reaction time optimization experiment for the porcine epidemic diarrhea virus G2c subtype MIRA-LFD; from left to right, the five test strips represent the reaction times at 42℃ for 5 min, 10 min, 15 min, 20 min, and 25 min, respectively; N is the negative control; Figure 4 This image shows the specificity test results for the MIRA-LFD subtype of porcine epidemic diarrhea virus (PEDV) G2c. From left to right, the test strips represent the negative control, porcine rotavirus (PoRV), porcine delta coronavirus (PDCoV), porcine transmissible gastroenteritis virus (TGEV), PEDV G2c subtype strain (PEDV / HK2021), PEDV G1b subtype strain (PEDV JS2008), PEDV S-INDEL subtype strain (PEDV NJ-32), and PEDV G2b subtype strain (PEDV AH2012 / 12). Figure 5 Figure showing the sensitivity test results for the porcine epidemic diarrhea virus G2c subtype MIRA-LFD; Figure 6 This image shows the results of clinical sample testing for the porcine epidemic diarrhea virus G2c subtype MIRA-LFD. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Unless otherwise specified, the operations in the following examples are all conventional experimental methods. Unless otherwise specified, the experimental materials in the following examples were all purchased from commercial channels.

[0025] The porcine epidemic diarrhea virus (PEDV) strains HK2021, JS2008, NJ-32, AH2012 / 12, rotavirus, porcine delta coronavirus, and transmissible gastroenteritis virus (TGEV) were all provided by the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences.

[0026] Example 1: Primer Design and Synthesis The S gene of porcine epidemic diarrhea virus (PEDV) plays a crucial role in viral evolution and is commonly used to distinguish different PEDV genotypes. Therefore, the S gene sequences of different PEDV genotypes were downloaded from the NCBI database, compared and analyzed using MEGA 7.0 software, and a phylogenetic tree was constructed. See [link to sequence comparison]. Figure 1 A pair of primers, namely the G2c upstream primer and the G2c downstream primer, were designed using PrimerExpress 3.0 software and synthesized by Sangon Biotech (Shanghai) Co., Ltd., as detailed in Table 1.

[0027] Table 1 Primer sequences for the G2c subtype of porcine epidemic diarrhea virus MIRA-LFD Note: "6-FAM" represents the 6-carboxyfluorescein group; "Biotin" represents the biotin group. The 3' end of the above G2c-F is set to the SNP base T of G2c (different from the corresponding base of other PEDV gene subtypes), and a mismatched base C is introduced at position 5 of the 3' end and a mismatched base A is introduced at position 9 of the 3' end to improve the amplification specificity of the G2c target sequence.

[0028] Example 2: Preparation of positive and negative controls (1) Determination of the toxic value of PEDV / HK2021 The PEDV / HK2021 strain was serially diluted 10-fold and inoculated into Vero cells for half-maximal tissue culture infectious dose (TCID). 50 ) Determination. TCID of PEDV / HK2021 was determined. 50 1×10 6 / mL.

[0029] (2) Preparation of positive control RNA was extracted from 500 μL of PEDV / HK2021 virus solution (virus titer: 1×10⁻⁶) using a rapid RNA extraction kit (Novizan Group, catalog number RC113-01). 6 TCID 50 RNA was extracted from the sample and used as a positive control, which was then stored at -80°C.

[0030] (3) Preparation of negative control The whole genome was extracted from Vero cells using a rapid RNA extraction kit (Novizan Group, catalog number RC113-01) as a negative control.

[0031] Example 3: Optimization of reaction temperature for porcine epidemic diarrhea virus G2c genotype MIRA-LFD Using the controlled single-variable method, MIRA was amplified for 20 min at reaction temperatures of 36℃, 38℃, 40℃, 42℃, 44℃, and 46℃, respectively, with a template of 1×10⁻⁶. 3.5 TCID 50 The HK2021 strain RNA template was used. After the reaction, the product was diluted 150-fold, and the results were observed using a lateral flow chromatography nucleic acid test strip.

[0032] The results showed that when the reaction temperature was between 36℃ and 42℃, the brightness of the test strip's detection line gradually increased. Figure 2 At 42℃-46℃, the brightness of the test strip detection lines gradually decreases. Figure 2 42℃ can be considered the optimal temperature for MIRA amplification.

[0033] Example 4: Optimization of reaction time for porcine epidemic diarrhea virus G2c genotype MIRA-LFD Using the controlled single-variable method, MIRA was amplified at a reaction temperature of 42℃ for 5 min, 10 min, 15 min, 20 min, and 25 min, respectively, with a template of 1×10⁻⁶. 3.5 TCID 50 The HK2021 strain RNA template was used. After the reaction, the product was diluted 150-fold, and the results were observed using a lateral flow chromatography nucleic acid test strip.

[0034] The results showed that when the reaction time was 15 minutes, the test strip's detection line was brighter. Figure 3 During the 15-25 minute interval, the brightness of the test strip detection line showed no significant change. Figure 3 ).

[0035] Example 5: Establishment of the MIRA-LFD method for porcine epidemic diarrhea virus G2c genotype 1. The single nucleic acid test strip (purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: B110202-48) consists of a sample pad, a conjugate pad, an NC membrane, and an absorbent pad. The NC membrane detection zone captures the amplification products of G2c-F and G2c-R (red band); the control zone (C) is the standard for determining whether the sample is sufficient and whether the chromatography process is normal. Regardless of whether the labeled nucleic acid amplification product is present in the sample, a blue band will appear in the control zone (C).

[0036] 2. The reaction system is as follows: 1 μL of G2c-F, the upstream primer of porcine epidemic diarrhea virus G2c, at a concentration of 10 μmol / L; 1 μL of G2c-R downstream primer of porcine epidemic diarrhea virus G2c at a concentration of 10 μmol / L; Take 12 μL of a single lyophilized microsphere dissolved in 30 μL Buffer A; Buffer B 1μL; 7 μL of sterile deionized water; 3 μL of RNA sample to be tested; The total amount of reagents in the reaction tube was 25 μL, which is the amount used for a single reaction.

[0037] 3. The reaction process is as follows: 1) Extract RNA from the sample to be tested: Extract genomic RNA from the sample to be tested as the template RNA for testing; 2) The amplification reaction system is as follows: 22 μL amplification reaction solution; 3 μL template RNA to be tested or positive control or negative control; the total volume of the reaction system is 25 μL; 3) Isothermal amplification of porcine epidemic diarrhea virus G2c gene subtype: The amplification reaction system prepared in step 2) was amplified in a constant temperature device under the following conditions: 42℃ for 15 min. 4) Detection of isothermal amplification-LFD results of porcine epidemic diarrhea virus G2c gene subtype: Dilute the amplification product in step 3) 150 times, and add 80 μL to the sample well of the lateral flow chromatography test strip; 5) Result determination: The test was successful under the following conditions: the positive control lateral flow chromatography test strip showed a red test line, the negative control lateral flow chromatography test strip showed no color development, and all lateral flow chromatography test strips showed a blue control line. A positive result is indicated by a red test line on the lateral flow chromatography test strip; a negative result is indicated by no color development on the test strip.

[0038] Example 6: Specificity analysis of the MIRA-LFD kit for porcine epidemic diarrhea virus G2c genotype. Specificity tests were conducted using the reaction system and reaction procedure of Example 5. The templates used were porcine epidemic diarrhea virus G2c subtype (HK2021), porcine epidemic diarrhea virus G1b subtype (JS2008), porcine epidemic diarrhea virus G2b subtype (AH2012 / 12), porcine epidemic diarrhea virus S-INDEL subtype (NJ-32), porcine rotavirus (PoRV), porcine delta coronavirus (PDCoV), porcine transmissible gastroenteritis virus (TGEV), and a negative control.

[0039] like Figure 4 As shown, the results indicated that no bands were detected for porcine rotavirus (PoRV), porcine delta coronavirus (PDCoV), porcine transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus G1b genotype (JS2008), porcine epidemic diarrhea virus S-INDEL genotype (NJ-32), porcine epidemic diarrhea virus G2b genotype (AH2012 / 12), and the negative control. Only porcine epidemic diarrhea virus G2c genotype (HK2021) showed a reactive band on the test line. All control lines showed normal color development.

[0040] Example 7: Sensitivity analysis of the MIRA-LFD kit for porcine epidemic diarrhea virus G2c genotype. 1×10 6 TCID 50 The PEDV / HK2021 strain virus fluid was diluted to a titer range of 1×10⁻⁶. 1 TCID 50 1×10 1.5 TCID 50 1×10 2TCID 50 1×10 2.5 TCID 50 RNA was extracted from 500 μL of each concentration, and the prepared RNA template was used to analyze sensitivity.

[0041] like Figure 5 As shown in the figure, the results indicate that the sensitivity of this kit is 1×10⁻⁶. 1.5 TCID 50 .

[0042] Example 8: Clinical sample detection using the MIRA-LFD kit for porcine epidemic diarrhea virus G2c genotype: RNA was extracted from 22 clinically collected fecal samples using a rapid RNA extraction kit (Novizan Group, catalog number RC113-01) and used as the template for MIRA-LFD detection. MIRA-LFD detection was performed using the reaction system and conditions described in Example 5. cDNA obtained from the RNA using a reverse transcription reagent (Kangwei Century, catalog number CW2020M) was used as the template for qPCR detection and was simultaneously detected using the PEDV gate qPCR method. The reaction system for the gate qPCR method was as follows: 0.4 μL each of 10 μmol / L upstream primer PEDV-F and downstream primer PEDV-R; 0.2 μL of 10 μmol / L probe PEDV-P; 10 μL of 2 × AceQ qPCR ProbeMaster Mix buffer; 0.4 μL of ROX Passive Reference Dye; 7.6 μL of sterile deionized water; and 1 μL of the nucleic acid template to be tested. Reaction conditions: Pre-denaturation at 95℃ for 5 min; 95℃ for 10 s; 60℃ for 30 s, 40 cycles; fluorescence signal acquisition at 60℃ for 30 s. Sterile deionized water was used as a blank control. Primer and probe sequences are shown in Table 2.

[0043] Table 2 Primer and probe sequences for qPCR detection of the N gene of porcine epidemic diarrhea virus. Eleven PEDV-positive samples were detected by qPCR using the N gene of porcine epidemic diarrhea virus (PEDV). Subsequent analysis using the S gene PCR, sequencing, and alignment process yielded genotyping information, with seven samples identifying the G2c genotype (see Table 3). MIRA-LFD analysis of the aforementioned 22 samples revealed four G2c-positive samples, all of which were verified as G2c by alignment analysis (see Table 3). Figure 6 MIRA-LFD can detect G2c samples with a Ct value below 33 by qPCR. Combined with the advantages of simple operation, immediacy, and no dependence on large instruments, this method has significant advantages in on-site detection of PEDV G2c gene subtypes in clinical settings and can meet the needs of rapid detection.

[0044] Table 3. Results of qPCR detection of the N gene of porcine epidemic diarrhea virus (PEDV) in 22 samples. Note: "NO" indicates a negative result for the qPCR test of the N gene of porcine epidemic diarrhea virus; Ct≥36 is negative, and Ct<36 is positive.

[0045] 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 primer combination for rapid detection of porcine epidemic diarrhea virus G2c gene subtype isothermal amplification-LFD, characterized in that, The primer combination consists of an upstream primer with nucleotide sequences as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.

2.

2. The primer combination as described in claim 1, characterized in that, The upstream primer is labeled with a 6-carboxyfluorescein group at its 5' end, and the downstream primer is labeled with a biotin group at its 5' end.

3. A rapid detection kit for porcine epidemic diarrhea virus G2c gene subtype isothermal amplification-LFD, characterized in that, Includes the primer combination described in claim 1 or 2.

4. The kit according to claim 3, characterized in that, The kit also includes: lyophilized powder, amplification buffer, sterile deionized water, lateral flow chromatography test strips, negative control, and positive control.

5. The use of the primer combination as described in claim 1 or 2 in the preparation of a product for detecting the G2c gene subtype of porcine epidemic diarrhea virus.

6. The application as described in claim 5, characterized in that, The detection method is as follows: using the genomic RNA of the porcine epidemic diarrhea virus sample to be tested as a template, isothermal amplification is performed using the upstream primer and the downstream primer. The amplification product is detected using a lateral flow chromatography test strip. Based on the detection result of the lateral flow chromatography test strip, it is determined whether the porcine epidemic diarrhea virus sample to be tested contains the porcine epidemic diarrhea virus G2c gene subtype.

7. The application as described in claim 6, characterized in that, The method for judgment is as follows: when the detection line of the lateral flow chromatography test strip turns red, it is judged as positive, that is, the porcine epidemic diarrhea virus sample to be tested contains the porcine epidemic diarrhea virus G2c gene subtype. When the test line of the lateral flow chromatography test strip shows no color development, it is judged as negative, that is, the porcine epidemic diarrhea virus sample to be tested does not contain the porcine epidemic diarrhea virus G2c gene subtype.

8. The use of the primer combination as described in claim 1 or 2 in the preparation of products for diagnosing infection with the G2c gene subtype of porcine epidemic diarrhea virus.

9. The application as described in claim 8, characterized in that, The diagnosis is as follows: using the genomic RNA of the sample to be tested as a template, isothermal amplification is performed using the upstream primer and the downstream primer, and the amplification product is detected using a lateral flow chromatography test strip. Based on the detection result of the lateral flow chromatography test strip, it is determined whether the sample to be tested is infected with the porcine epidemic diarrhea virus G2c gene subtype.

10. The application as described in claim 9, characterized in that, The method for judgment is as follows: when the detection line of the lateral flow chromatography test strip turns red, it is judged as positive, that is, the sample to be tested is a sample infected with the G2c gene subtype of porcine epidemic diarrhea virus. When the test line of the lateral flow chromatography test strip shows no color development, it is judged as negative, that is, the sample to be tested is a sample that has not been infected by the porcine epidemic diarrhea virus G2c gene subtype.

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