Kit for identifying G2b gene subtype of porcine epidemic diarrhea virus and application

Abstract

The invention discloses a kit for identifying a G2b gene subtype of a porcine epidemic diarrhea virus and application of the kit, and belongs to the field of virus nucleic acid detection. Through gene comparison, primers and probes are designed at specific sequence positions of the PEDV G2b gene subtype, and quantitative PCR identification detection can be directly carried out through an LNA technology. The detection method only comprises two steps of nucleic acid extraction and one-step fluorescent RT-PCR reaction, can complete identification within 2 hours at the soonest, has the characteristics of good specificity, high sensitivity and good stability, can rapidly, simply, conveniently and efficiently identify and detect PEDV G2b subtype nucleic acid, is low in cost and short in period, is especially suitable for high-throughput detection, and can be widely applied to detection of PEDV G2b subtype nucleic acid. And the method has important significance on rapid identification of PEDV G2b gene subtype strains.

Description

Technical Field

[0001] This invention relates to the field of viral nucleic acid detection, and in particular to a kit for identifying the G2b gene subtype of porcine epidemic diarrhea virus and its application. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded, positive-sense RNA virus belonging to the family Coronaviridae and the genus Alphacoronavirus. Clinically, it primarily causes diarrheal symptoms in sows and piglets, characterized by vomiting, watery diarrhea, dehydration, and decreased appetite. In 2010, a novel PEDV mutant strain (G2) emerged, resulting in a near 100% morbidity rate and a mortality rate of approximately 80%–100% in piglets under 7 days old.

[0003] PEDV genotyping, after alignment of the full-length S gene sequence, can be divided into three main genotypes: G1 type (including two subtypes, G1a and G1b), G2 type (including three subtypes, G2a, G2b, and G2c), and S-INDEL type. Currently, only full-length S gene sequencing can differentiate the genotype of the strain. This method involves steps such as nucleic acid extraction, RNA reverse transcription, PCR amplification, agarose gel electrophoresis, gel cutting, sequencing, splicing the sequencing results to obtain the full-length S gene sequence, alignment with MEGA software, and construction of a phylogenetic tree. It typically takes 1-2 days to complete genotyping, which is time-consuming and cannot promptly identify PEDV genotypes.

[0004] Locked nucleic acid (LNA) is a synthetic nucleic acid analog containing bridging bicyclic glycosyl groups. By incorporating LNA-modified nucleotides into primers and / or probes, the ability of quantitative PCR primers and / or probes to distinguish mutant genes by single nucleotide polymorphisms (SNPs) is greatly enhanced due to LNA's strong tendency to form mismatched Watson-Crick base pairs. However, there are currently no reports of using LNA technology to distinguish PEDV gene subtypes. Summary of the Invention

[0005] The purpose of this invention is to provide a kit and its application for identifying the G2b gene subtype of porcine epidemic diarrhea virus (PEDV) to solve the problems existing in the prior art. The kit has good specificity, high sensitivity, and good stability, and can quickly, easily, and efficiently identify PEDV G2b subtype nucleic acid. It is low in cost, has a short cycle time, and is particularly suitable for high-throughput detection.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a primer-probe combination for the G2b gene subtype of porcine epidemic diarrhea virus, wherein the primer-probe combination is as follows:

[0008] Upstream primer: 5'-TGCTTTTGACCTTGACGATGG-3' (SEQ ID NO.1);

[0009] Downstream primer: 5'-TGTCAGATGCAATaAGGTTGG-3' (SEQ ID NO.2);

[0010] Probe: 5'-TTAAtATTACTGTCTCTGCtGC-3' (SEQ ID NO.3);

[0011] In the nucleotide sequence of the probe, lowercase letter bases are LNA-modified bases.

[0012] Preferably, the 5' end of the probe is modified with a fluorescent group, and the 3' end is modified with a quenching group.

[0013] The present invention also provides the application of the primer-probe combination in the preparation of a kit for identifying the G2b gene subtype of porcine epidemic diarrhea virus.

[0014] The present invention also provides the application of the primer-probe combination in the preparation of a kit for diagnosing infection of the porcine epidemic diarrhea virus G2b gene subtype.

[0015] The present invention also provides a kit for identifying the G2b gene subtype of porcine epidemic diarrhea virus, comprising the aforementioned primer and probe combination.

[0016] Preferably, when amplifying a porcine epidemic diarrhea virus sample to be identified using the primer-probe combination, the amplification system includes the following components in the following amounts: 5× buffer 5.0 µL, enzyme reaction solution 1.25 µL, upstream primer and downstream primer 1.0 µL each, probe 0.5 µL, signal correction solution 0.5 µL, RNA template 5.0 µL, and nuclease-free water 10.75 µL.

[0017] Preferably, the amplification program is as follows: reverse transcription at 55°C for 15 min, 1 cycle; pre-denaturation at 95°C for 30 s, 1 cycle; denaturation at 95°C for 10 s, annealing at 60°C for 45 s, 40 cycles; and the fluorescence signal of the amplification curve is automatically collected at the end of each amplification cycle.

[0018] Preferably, the presence of the G2b genotype of porcine epidemic diarrhea virus in the sample to be identified is determined based on the collected fluorescence signal.

[0019] The criteria for judgment are as follows: if there is a clear amplification curve and the Ct value is <37, the porcine epidemic diarrhea virus sample to be identified is judged as positive; if there is no amplification curve or no Ct value, the porcine epidemic diarrhea virus sample to be identified is judged as negative; if there is a clear amplification curve and the Ct value is between 37 and 40, the porcine epidemic diarrhea virus sample to be identified needs to be tested repeatedly, and if the Ct value of the repeated test is between 37 and 40, it is judged as positive.

[0020] Preferably, the kit also includes a positive control and a negative control.

[0021] The present invention discloses the following technical effects:

[0022] (1) High specificity: The kit of the present invention contains a specially designed primer and probe combination. This primer and probe combination can only specifically detect PEDV G2b gene subtype strains and cannot detect other PEDV gene subtypes.

[0023] (2) High sensitivity: The kit of the present invention can detect as low as 1 TCID. 50 / mL virus.

[0024] (3) Good reproducibility: The kit of the present invention has good intra-batch and inter-batch reproducibility, with intra-batch and inter-batch coefficients of variation of less than 1% and 3%, respectively.

[0025] (4) Simple and fast: By comparing genes, primers and probes are designed at the sequence positions specific to the PEDV G2b gene subtype, and quantitative PCR identification can be performed directly by LNA technology. This detection method only has two steps: nucleic acid extraction and one-step fluorescent RT-PCR reaction. It is simple to operate and fast to run, and identification can be completed in as little as 2 hours.

[0026] Therefore, the kit of the present invention provides a technical means for the identification and detection of PEDV G2b gene subtypes, which is of great significance for the clinical diagnosis and prevention of PEDV G2b gene subtype infection. Attached Figure Description

[0027] 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.

[0028] Figure 1 The amplification curve of PEDV G2b gene subtype using one-step LNA-TaqMan probe fluorescence RT-PCR is shown.

[0029] Figure 2Standard curve for one-step LNA-TaqMan probe fluorescence RT-PCR of PEDV G2b gene subtype;

[0030] Figure 3 This is a one-step LNA-TaqMan probe fluorescent RT-PCR specificity assay for PEDV G2b genotype; where 1 is the PEDV G2b positive control, and 2-12 are the PEDV G1b genotype, PEDV G2c genotype, PEDV S-INDEL genotype, PoRVA, TGEV, PDCoV, GETV, PSV, PTV, PoNoV and negative control, respectively.

[0031] Figure 4 Sensitivity assay of one-step LNA-TaqMan probe fluorescent RT-PCR for PEDV G2b gene subtype. Detailed Implementation

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] Example 1: Design and synthesis of primers and probes for PEDV G2b gene subtype

[0038] Download the strain sequences of porcine epidemic diarrhea virus (PEDV) from NCBI, including G1a (GU937797, JQ023161, JQ239433, LT906620), G1b (KC109141, KC210146, KT323979), G2a (JN825712, KC210145, MH053419, MH061337, MH061342, MH243316, MK690502, MN893425, OQ412638), and G2b (JX188454, KF384500, KT021231, KT02123). 3. The S gene sequences of KU646831, KU985230, KY4963150, MH748550, OQ269588, OR418363, G2c (KM609212, KU252649, MF375374, OL762457, ON155919, OQ412639) and S-INDEL (MH593148, MH061336, KU847996, KJ399978, OR608353) were compared using SnapGene 6.0 software. Primers and probes were designed at the G2b genotype subtype-specific sequence positions. All primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0039] The primer and probe sequences are shown below:

[0040] Upstream primer (PEDV-SF) sequence: 5'-TGCTTTTGACCTTGACGATGG-3' (SEQ ID NO.1);

[0041] Downstream primer (PEDV-SR) sequence: 5'-TGTCAGATGCAATaAGGTTGG-3' (SEQ ID NO.2);

[0042] Probe (G2b-S-probe) sequence: 5'-FAM-TTAAtATTACTGTCTCTGCtGC-BHQ1-3' (SEQ ID NO.3).

[0043] In the probe sequence above, lowercase letters are LNA-modified bases.

[0044] Example 2: Extraction and dilution of PEDV AH2012 / 12 viral nucleic acid

[0045] Take 10 6.5 TCID 50 10 mL of PEDV AH2012 / 12 cell culture was used to extract total RNA using a viral DNA / RNA extraction kit (ONREW, China). RNA was eluted with 3 mL of RNA storage solution (ThermoFisher Scientific, USA) and stored at -80°C for later use. After concentration, the RNA concentration at this point corresponds to a viral concentration of 10⁻⁶. 7.0 TCID 50 / mL, which is the standard positive nucleic acid. Then, using EASY Dilution (Takara, China), the standard positive nucleic acid is serially diluted 10-fold to obtain the final RNA concentration equivalent to a viral titer of 10. 6.0 Up to 10 0.1 TCID 50 / mL of nucleic acid solution.

[0046] Example 3: Optimization of primer and probe concentrations

[0047] Four different concentrations of primer and probe combinations (concentration A: 0.6 / 0.6 / 0.3, concentration B: 0.8 / 0.8 / 0.4, concentration C: 1.0 / 1.0 / 0.5, concentration D: 1.2 / 1.2 / 0.6) were added to the fluorescent RT-PCR reaction system, and 10... 4.0 TCID 50 Positive nucleic acid at a dilution of / mL was used as a template for simultaneous amplification. The signal intensity and Ct value of the amplification curves were compared to determine the optimal primer and probe concentrations. A comprehensive comparison determined concentration C to be the optimal combination (Table 1).

[0048] Table 1 Optimization of optimal primer and probe concentrations

[0049]

[0050] Example 4: Establishment of a one-step fluorescent RT-PCR standard curve for PEDV G2b genotype

[0051] All one-step fluorescent RT-PCR assays were performed using an ABI 7500 quantitative PCR instrument (Thermo Fisher Scientific, USA). Optimized reaction conditions were determined as follows: 5.0 µL 5× buffer (Vazyme, China), 1.25 µL enzyme reaction solution (Vazyme, China), 1.0 µL each of upstream and downstream primers (10 µM), 0.5 µL probe (10 µM), 0.5 µL ROX2 (signal correction solution), 5.0 µL RNA template, and 10.75 µL nuclease-free water, for a total reaction volume of 25.0 µL. The amplification procedure was as follows: reverse transcription at 55°C for 15 min, one cycle; pre-denaturation at 95°C for 30 s, one cycle; denaturation at 95°C for 10 s, annealing at 60°C for 45 s (fluorescence signal reading), 40 cycles. Fluorescence signals were automatically collected at the end of each amplification cycle. Based on the optimized reaction system and procedure, 10-fold serially diluted viral positive RNA templates (concentration range 10) were analyzed. 6.0 ~ 10 1.0 TCID 50 Amplification was performed at ( / mL) to establish a standard curve, which was derived through linear regression analysis between the cycle threshold (Ct) value and the logarithm of the viral RNA concentration. The amplification curve was good ( Figure 1 The standard curve equation is Y = -3.449X + 36.511, the amplification efficiency is E = 94.946%, and the correlation coefficient is R. 2 =0.995 ( Figure 2 ).

[0052] Example 5: Specificity Test

[0053] The following porcine viral nucleic acid templates preserved in the laboratory were tested using the test kit of this invention: porcine epidemic diarrhea virus (PEDV) G1b genotype, PEDV G2c genotype, PEDV S-INDEL genotype, porcine rotavirus group A (PoRVA), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), getta virus (GETV), porcine sapellovirus (PSV), porcine chezovirus (PTV), and porcine norovirus (PoNoV). Figure 3 Marker 2-11 in the text); at the same time, PEDV G2b gene subtype nucleic acid was set as a positive control ( Figure 3 Marker 1), nuclease-free water as a negative control ( Figure 3 (Mark 12 in the text). The fluorescent RT-PCR reaction system and reaction procedure are the same as in Example 4.

[0054] The results showed that only the positive control exhibited a typical amplification curve, while the others showed no amplification, indicating that the fluorescent RT-PCR primers and probes of this invention have good specificity. Figure 3 ).

[0055] Example 6: Sensitivity Test

[0056] The standard positive nucleic acid was diluted 10 times (10 6.0 ~10 0.1 TCID 50 Amplification was performed using the kit of this invention ( / mL), and the results showed that 10 6.0 ~1 TCID 50 The kit showed clear amplification curves at / mL, indicating that the limit of detection (LOD) of this kit is 1 TCID. 50 / mL, with good sensitivity ( Figure 4 ).

[0057] Example 7: Repeatability Test

[0058] The reproducibility of fluorescent RT-PCR assays was assessed by calculating intra- and inter-assay coefficients of variation (CV). Using 10... 6.0 10 4.0 10 2.0 TCID 50 Standard RNA of the PEDV G2b gene subtype was used as a template at / mL. For intra-batch reproducibility, each template was tested three times in a single experimental run. For inter-batch reproducibility, each template was analyzed in three independent experiments conducted on different dates. The results showed that the coefficients of variation (CV) of the intra-batch and inter-batch Ct values ​​were less than 1% (0.54%–0.74%) and 3% (1.10%–2.35%), respectively (Table 2).

[0059] Table 2 Repeatability Tests

[0060]

[0061] Example 8: Clinical Sample Testing

[0062] Ten PEDV-positive fecal samples (samples 1-10) and five PEDV-negative fecal samples (samples 11-15) were collected from clinical animals, including one sample of G1b, three samples of G2b, five samples of G2c, one sample of S-INDEL, and five samples of artificially mixed nucleic acids (samples 16-20). The feces were resuspended in PBS, vortexed, centrifuged, and the supernatant was collected. Viral nucleic acid was extracted using a viral RNA extraction kit (RC112-01, Novizan). Amplification was performed using the fluorescent RT-PCR primers and probes of this invention, with the fluorescent RT-PCR reaction system and procedure the same as in Example 4. This kit requires both a positive and a negative control for each sample test. The test is valid when the positive control shows effective amplification and the negative control shows no amplification. The criteria for judging the sample results are as follows: if there is a clear amplification curve and the Ct value is <37, the sample is considered positive; if there is no amplification curve or no Ct value, the sample is considered negative; if there is a clear amplification curve and the Ct value is between 37 and 40, the sample needs to be tested again. If the Ct value of the repeated test is still within this range, the sample is considered positive.

[0063] The results showed that samples containing only the G2b gene subtype nucleic acid were positive; all other samples were negative (Table 3).

[0064] Table 3 Clinical Sample Test Results

[0065]

[0066] 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 probe combination of a G2b gene subtype of porcine epidemic diarrhea virus, characterized by, The primer probe combination is: an upstream primer: 5'-TGCTTTTGACCTTGACGATGG-3'; a downstream primer: 5'-TGTCAGATGCAATaAGGTTGG-3'; a probe: 5'-TTAAtATTACTGTCTCTGCtGC-3'; wherein, in the nucleotide sequence of the probe, the lowercase base is an LNA modified base.

2. The primer probe combination of claim 1, wherein, The 5' end of the probe is modified with a fluorescent group, and the 3' end is modified with a quenching group.

3. Use of the primer probe combination of claim 1 or 2 in the preparation of a kit for identifying the G2b gene subtype of porcine epidemic diarrhea virus.

4. Use of the primer probe combination of claim 1 or 2 in the preparation of a kit for diagnosing infection with the G2b gene subtype of porcine epidemic diarrhea virus.

5. A kit for identifying the G2b gene subtypes of porcine epidemic diarrhea virus, characterized in that, The kit comprises the primer probe combination of claim 1 or 2.

6. The kit of claim 5, wherein When amplifying a sample of porcine epidemic diarrhea virus to be identified using the primer probe combination, the following components are included in the amplification system in the following amounts: 5x buffer 5.0 µL, enzyme reaction solution 1.25 µL, upstream primer and downstream primer each 1.0 µL, probe 0.5 µL, signal correction solution 0.5 µL, RNA template 5.0 µL, and nuclease-free water 10.75 µL.

7. The kit of claim 6, wherein The amplification program is: reverse transcription at 55°C for 15 min, 1 cycle; pre-denaturation at 95°C for 30 s, 1 cycle; denaturation at 95°C for 10 s and annealing at 60°C for 45 s, 40 cycles; automatic collection of amplification curve fluorescence signals at the end of each amplification cycle.

8. The kit of claim 7, wherein According to the collected fluorescence signals, it is determined whether the sample of porcine epidemic diarrhea virus to be identified contains the G2b gene subtype of porcine epidemic diarrhea virus; The determination standard is: if there is a clear amplification curve and the Ct value is < 37, then the sample of porcine epidemic diarrhea virus to be identified is determined to be positive; if there is no amplification curve or no Ct value, then the sample of porcine epidemic diarrhea virus to be identified is determined to be negative; if there is a clear amplification curve and the Ct value is between 37 and 40, the sample of porcine epidemic diarrhea virus to be identified needs to be retested, and if the Ct value of the repeated test is between 37 and 40, it is determined to be positive.

9. The kit of claim 5, wherein The kit further comprises positive and negative controls.

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