RT-qPCR (real-time quantitative polymerase chain reaction) detection primer group for multi-host coronavirus D nucleic acid and application thereof

Abstract

The invention discloses an RT-qPCR (real-time quantitative polymerase chain reaction) detection primer group for multi-host coronavirus D nucleic acid and application of the RT-qPCR detection primer group. The detection primer group comprises a pair of primer pairs DCoV-N-F / R and a probe, the primer group developed by the invention has the advantages of strong specificity, high sensitivity and good repeatability in detection, and meanwhile, the RT-qPCR detection kit for the coronavirus delta is constructed by optimizing detection conditions such as a probe design strategy and a reaction system, so that accurate screening of the coronavirus delta in wild birds and livestock groups is realized; and a good technical support is provided for diagnosis of the coronavirus delta. And a technical support is provided for analyzing a virus evolution rule and formulating a prevention and control strategy.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnosis technology, and particularly relates to an RT-qPCR detection primer set for nucleic acid of multi-host deltacoronavirus and its application. Background Art

[0002] Coronaviruses belong to the subfamily Orthocoronavirinae of the family Coronaviridae, and are a class of single-stranded positive-sense RNA viruses with an envelope structure. Their genome lengths reach 26 - 32 kb and can be divided into four genera: Alpha, Beta, Gamma, and Delta. The virus particles contain four key structural proteins: the spike protein (S) mediates host cell receptor binding and membrane fusion, the membrane protein (M) participates in virus assembly, the envelope protein (E) regulates virus budding, and the nucleocapsid protein (N) binds to viral RNA to form a helical nucleocapsid.

[0003] Among the deltacoronaviruses discovered in recent years, porcine deltacoronavirus (PDCoV), as a novel porcine enteropathogen, can cause high mortality in neonatal piglets through watery diarrhea, resulting in serious economic losses. Traceability studies have shown that PDCoV may originate from deltacoronaviruses in wild bird populations. However, wild bird deltacoronaviruses have a wide host distribution, significant genetic diversity, and complex recombination characteristics. Due to insufficient conservatism in primer design, existing detection methods are difficult to cover virus variants from different host sources, resulting in biases in monitoring data and severely restricting the research on the cross-host transmission mechanism and phylogenetic relationship of the virus.

[0004] There is an urgent need to establish a new universal molecular diagnosis technology for deltacoronavirus suitable for different hosts. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art, and provides an RT-qPCR detection primer set for nucleic acid of multi-host deltacoronavirus and its application. The primer set developed by the present invention has strong specificity, high sensitivity, and good repeatability in detection. At the same time, by optimizing detection conditions such as probe design strategies and reaction systems, an RT-qPCR detection kit for deltacoronavirus is constructed to achieve accurate screening of deltacoronavirus in wild bird and livestock populations, providing good technical support for the diagnosis of deltacoronavirus. It provides technical support for analyzing the virus evolution law and formulating prevention and control strategies.

[0006] To achieve the above purpose, the technical solutions designed by the present invention are as follows:

[0007] The present invention provides an RT-qPCR detection primer set for nucleic acid of multi-host deltacoronavirus, and the detection primer set includes a pair of primer pairs DCoV-N-F / R and 1 probe; among them, the primer pair RT-F / R is as follows:

[0008] DCoV-N-F: 5'-CAGGTKCTYAARGCTCA-3',

[0009] DCoV-N-R: 5'-AGGMAWAGGATGGAGGAA-3';

[0010] The probe sequence is: 5'-CCATGGCTAYTGGCTVCGTTWCA-3'.

[0011] Furthermore, the 5' end of the probe is labeled with a HEX group and the 3' end is labeled with a BHQ1 group.

[0012] The present invention also provides the use of the above RT-qPCR detection primer set in the preparation of an RT-qPCR detection kit for multi-host deltacoronavirus.

[0013] The present invention also provides an RT-qPCR detection kit for multi-host deltacoronavirus, and the kit includes an RT-qPCR detection primer set.

[0014] Furthermore, the RT-qPCR detection kit further includes 2×PerfectStartTM Probe One-step qPCR SuperMix, TransScript Probe One-step RT / RI Enzyme Mix, and RNase-free Water.

[0015] The present invention also provides the use of the above RT-qPCR detection kit in the detection of deltacoronavirus nucleic acid.

[0016] The present invention also provides a method for detecting multi-host deltacoronavirus nucleic acid, comprising the following steps:

[0017] 1) Extract DNA from the sample to be tested and then reverse transcribe the RNA;

[0018] 2) Using the above RNA as a template, perform RT-qPCR with the above kit to obtain the Ct value;

[0019] 3) According to the standard curve in the kit, use the Ct value for comparison and interpretation to judge:

[0020] When the Ct value < 37, the detection of deltacoronavirus nucleic acid in the sample to be tested is positive;

[0021] When the Ct value ≥ 37, the detection of deltacoronavirus nucleic acid in the sample to be tested is negative.

[0022] Further, in the step 3), the reaction system for RT-qPCR is a 10 μL reaction system, including 2×PerfectStartTM Probe One-step qPCR SuperMix, TransScript Probe One-step RT / RI Enzyme Mix, upstream primer, downstream primer, probe, template and RNase-free Water;

[0023] The final concentrations of the upstream primer and the downstream primer in the reaction system are both 0.5 μmol / L, and the final concentration of the probe in the reaction system is 0.2 μmol / L.

[0024] Still further, in the step 3), the reaction conditions for RT-qPCR are 45 °C for 5 minutes, 94 °C for 30 seconds, followed by 45 cycles, each cycle being 94 °C for 5 seconds and 56 °C for 30 seconds.

[0025] Advantages of the present invention:

[0026] The detection kit for hepatitis D coronavirus provided by the present invention uses the RT-qPCR method to detect hepatitis D coronavirus. The kit has strong specificity, good repeatability and high sensitivity for the detection of hepatitis D coronavirus, and its lowest detection concentration can reach 10 copies / μL. Description of the drawings

[0027] Figure 1 It is the agarose gel electrophoresis diagram of the standard plasmid constructed in the example;

[0028] Figure 2 It is the result diagram of the ordinary PCR verification of the RT-qPCR primer in Example 2;

[0029] Figure 3 It is the result diagram of the reaction system optimization experiment in Example 2;

[0030] Figure 4 It is the result diagram of the reaction program optimization experiment in Example 2;

[0031] Figure 5 It is the RT-qPCR amplification curve diagram of different concentrations of standard plasmids in Example 2; 1-7 are 5×10 1 -5×10 7 copies / μL;

[0032] Figure 6 It is the RT-qPCR standard curve diagram established in Example 2;

[0033] Figure 7Results graph of the sensitivity experiment of RT-qPCR in Example 3; 1-6 are respectively 5×10 0 -5×10 5 copies / μL;

[0034] Figure 8 Results graph of the specificity experiment in Example 4;

[0035] Figure 9 Results graph of the RT-qPCR compatibility detection of deltacoronavirus and influenza A virus;

[0036] Figure 10 Partial detection results graph of RT-qPCR;

[0037] Figure 11 Results graph of the detection by the method of deltacoronavirus of swine SN / T 5124-2019. Specific implementation manners

[0038] The present invention will be further described in detail below in conjunction with specific embodiments for those skilled in the art to understand.

[0039] Example 1 Construction of standard plasmid

[0040] Select the relatively conserved N gene to construct the standard plasmid. First, amplify a partial fragment of the N gene, and the primer sequences for amplification are shown in Table 1.

[0041] Table 1 Primers for amplifying N gene fragment

[0042]

[0043] Extract the genomic RNA of sparrow deltacoronavirus spDCoV by a conventional RNA extraction method and perform reverse transcription. Use the obtained cDNA as a template for PCR reaction. The PCR reaction system and reaction program are as follows:

[0044] The 25 μL reaction system for the PCR reaction is: 1.25 μL of 10 μM upstream primer, 1.25 μL of 10 μM downstream primer, 5 μL of 5×Q5 Reaction Buffer (NEB, catalog number M0491S), 0.5 μL of 10 μM dNTPs (NEB, catalog number M0491S), 0.25 μL of Q5 High-Fidelity DNA Polymerase (NEB, catalog number M0491S), 2 μL of genomic cDNA, 14.75 μL of ddH2O.

[0045] The PCR reaction program is: pre-denaturation at 98°C for 30 seconds, then perform 35 cycles, each cycle being denaturation at 98°C for 10 seconds, annealing at 54°C for 30 seconds, extension at 72°C for 1 minute, and extension at 72°C for 5 minutes after the cycle ends.

[0046] After the PCR reaction, the amplified product was subjected to 1% agarose gel electrophoresis at a voltage of 150 V for 20 minutes. The result was determined according to the size of the amplified band. When the amplified band was approximately 1051 bp, it indicated that a partial sequence of the N gene of spDCoV was obtained.

[0047] Gel extraction was carried out by a conventional method.

[0048] The amplified fragment was ligated to the pCE3Blunt vector (length 1798 bp, Vazyme, catalog number C603) by a conventional method to obtain a standard plasmid (length 2849 bp). The result of agarose gel electrophoresis was as Figure 1 shown. The sequencing was correct by Wuhan Hecai Gene Technology Co., Ltd. The concentration of the standard plasmid was 103.1 ng / μL, and the calculated copy number was 3.3×10 10 copies / μL. A standard plasmid mother solution with a copy number of 5×10 9 copies / μL was prepared and named DCoV_stdN.

[0049] Example 2 Establishment of primer design for one-step reverse transcription real-time fluorescence PCR (RT-qPCR)

[0050] Probes were designed according to the conserved domain of the N gene. Several pairs of primers were designed based on the probes and primer screening was carried out. The optimal primer and probe sequences are shown in Table 2.

[0051] The RT-qPCR primers were verified by ordinary PCR. The obtained PCR product was the target sequence with a length of 160 bp. The verification result of agarose gel electrophoresis was as Figure 2 shown. The RT-qPCR primers had specific specificity.

[0052] Table 2 RT-qPCR primer and probe sequences

[0053]

[0054] Example 3 Establishment of an RT-qPCR detection kit for multi-host deltacoronavirus and method optimization

[0055] The RT-qPCR detection kit for multi-host deltacoronavirus includes the RT-qPCR detection primer set designed in Example 2, 2×PerfectStartTM Probe One-step qPCR SuperMix, TransScript Probe One-step RT / RI Enzyme Mix, and RNase-free Water.

[0056] I. Optimization of the detection method of the above RT-qPCR detection kit

[0057] 1. Experimental instruments and materials

[0058] The instrument used was a CFX Connect (Bio-Rad) qCPR instrument, and the reagent enzyme used was TransScript Probe One-Step qRT-PCR SuperMix purchased from TransGen Biotech, which contains 2×Perfect StartTM Probe One-step qPCR SuperMix (abbreviated as 2×qPCR SuperMix), TransScript Probe One-step RT / RI Enzyme Mix (abbreviated as RT / RI Enzyme Mix), and RNase-free Water.

[0059] To improve the sensitivity of the reaction, the reaction system, that is, the use concentrations of the probe and primers, was first determined. Both the probe and primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and were in dry powder form. They were diluted to 10 μmol / L with RNase-free Water as the stock solution.

[0060] The probe was diluted to 2 μmol / L, 4 μmol / L, 6 μmol / L, 8 μmol / L, and 10 μmol / L respectively with RNase-free Water.

[0061] The primers were diluted to 2 μmol / L, 4 μmol / L, 6 μmol / L, 8 μmol / L, and 10 μmol / L respectively with RNase-free Water.

[0062] 2. Optimize the reaction system

[0063] Using the RNA of avian-origin deltacoronavirus as the template, with the primers and probe shown in Table 2, the reaction program was: 45°C / 5 minutes, 94°C / 30 seconds, followed by 45 cycles, each cycle being 94°C / 5 seconds, 55°C / 30 seconds. RT-qPCR detections were carried out according to the reaction systems shown in Table 3 respectively:

[0064] Table 3 Reaction systems used in the screening experiment

[0065]

[0066] The reaction results are as Figure 3As shown, the curve marked by the red rhombus has a probe concentration of 4 μmol / L and upstream and downstream primer concentrations of 10 μmol / L. The Ct value of this curve is small, the sensitivity is high, and the amplification efficiency is high. Therefore, the primers and probes at this concentration were selected as the final system, that is, in the final reaction system, the final concentration of the upstream and downstream primers is 0.5 μmol / L, and the final concentration of the probe is 0.2 μmol / L. The finally optimized reaction system is shown in Table 4.

[0067] Table 4 Optimized reaction system

[0068]

[0069] 3. Optimization of annealing temperature

[0070] This experiment was mainly to determine the annealing temperature, which has a great influence on amplification.

[0071] Using the RNA of avian-origin deltacoronavirus as a template, with the primers and probes shown in Table 2, and the reaction system shown in Table 5, the reaction program was based on Table 4, and the annealing temperatures were set at 52 °C, 54 °C, 56 °C, 58 °C, and 60 °C respectively for RT-qPCR reactions. Two replicates were performed for each temperature.

[0072] Figure 4 The results showed that when the annealing temperature was 56 °C, the amplification efficiency and sensitivity were the highest. Therefore, this temperature was selected as the final annealing temperature, that is, the finally optimized reaction program was: 45 °C / 5 minutes, 94 °C / 30 seconds, followed by 45 cycles, each cycle being 94 °C / 5 seconds, 56 °C / 30 seconds.

[0073] 4. Establishment of standard curve

[0074] The standard plasmid was diluted to 5×10 1 -5×10 7 copies / μL using RNase-free Water, and RT-qPCR detection was performed using the primers and probes shown in Table 2. The reaction system was as shown in Table 4, and the reaction program was:

[0075] 45 °C / 5 minutes, 94 °C / 30 seconds, followed by 45 cycles, each cycle being 94 °C / 5 seconds, 56 °C / 30 seconds.

[0076] The amplification results are as Figure 5 shown. According to the amplification results, a standard curve was established between the Ct value and the logarithm of the standard product concentration, as Figure 6 shown. The standard curve equation was: Y = -3.513X + 44.49, R 2 = 0.998. From the standard curve equation, it can be seen that different concentration gradients are linearly correlated with the Ct value, and R 2Greater than 0.99, with good correlation.

[0077] From the amplification curves, it can be seen that the curves in the exponential growth phase are parallel, reflecting similar PCR amplification efficiencies. The Ct values vary evenly among different dilutions, and there is a good linear relationship between the Ct values and the copy numbers.

[0078] Based on the above optimization, the method for detecting the nucleic acid of multiple-host deltacoronavirus includes the following steps:

[0079] 1) Extract DNA from the sample to be tested and then reverse transcribe it into RNA;

[0080] 2) Using the above RNA as a template, perform RT-qPCR with the above kit to obtain Ct values;

[0081] 3) According to the standard curve in the kit, use the Ct values for comparative interpretation and judgment:

[0082] When the Ct value < 37, the nucleic acid detection of deltacoronavirus in the sample to be tested is positive;

[0083] When the Ct value ≥ 37, the nucleic acid detection of deltacoronavirus in the sample to be tested is negative.

[0084] II. Verification and analysis of the specificity, repeatability, and compatibility of the above kit

[0085] 1. Sensitivity verification and analysis

[0086] Dilute the standard plasmid to 5×10 0 -5×10 5 copies / μL using RNase-free Water, and perform sensitivity verification with the above kit.

[0087] The amplification results are as Figure 7 shown: When the Ct value is less than 37, the result is determined to be positive; when the Ct value is greater than or equal to 37, the result is determined to be negative, indicating good sensitivity of the detection method of this kit.

[0088] 2. Specificity verification and analysis

[0089] Select spDCoV (sparrow deltacoronavirus), PDCoV (porcine deltacoronavirus), PEDV (porcine epidemic diarrhea virus), TGEV (transmissible gastroenteritis virus), and IBV (infectious bronchitis virus), and perform specificity experiments with the above kit. All viral genomes are extracted using the conventional genomic RNA extraction method.

[0090] The experimental results are as Figure 8Shown as follows: Only spDCoV and PDCoV of the genus Deltacoronavirus had specific amplification, and no amplification was observed in other virus genera, indicating that the detection method of this kit has good specificity.

[0091] 3. Repeatability verification analysis

[0092] Dilute the standard plasmid to 5×10 3 -5×10 7 copies / μL with RNase-free Water and perform a repeatability experiment using the above kit.

[0093] The experimental results are shown in Table 5. The results show that the coefficient of variation of inter-group and intra-group repeats is less than 2.00%, indicating good repeatability.

[0094] Table 5 RT-qPCR repeatability verification

[0095]

[0096] 4. Compatibility analysis of the kit with the influenza virus detection method

[0097] Dilute the standard plasmid to 5×10 1 to 5×10 7 copies / μL with RNase-free Water and perform RT-qPCR detection using the above kit in combination with the Influenza A (Gene Matrix) CDC protocol2009 RT-qPCR detection primer set published by WHO. The reaction system is shown in Table 6, and the reaction program is as follows:

[0098] 45°C / 5 minutes, 94°C / 30 seconds, and then perform 45 cycles, each cycle being 94°C / 5 seconds, 56°C / 30 seconds.

[0099] The experimental results are shown in Figure 9 , indicating that the two methods are compatible in the same system.

[0100] Table 6 Dual RT-qPCR reaction system

[0101]

[0102] Example 4 Detection of clinical samples with the above kit

[0103] Collect samples from different regions for detection. Genomic DNA was extracted and reverse transcribed using conventional methods for all samples, and RT-qPCR detection was performed using the above kit and the porcine deltacoronavirus detection method published in the national standard SN / T 5124-2019, respectively.

[0104] The experimental results are as shown in Figure 10As shown in Table 10: The kit constructed by the present invention has a good detection effect on hepatitis D coronaviruses from multiple host sources.

[0105] Table 9 Primer sequences of SN / T 5124-2019

[0106]

[0107] Table 10 Detection results of clinical samples

[0108]

[0109] Note: *Wild birds include sparrows, coots, whooper swans, rock doves, black-headed gulls, common cormorants, Indian spot-billed ducks, grey herons, reef herons and pond herons. #This is the number of positive hepatitis D coronavirus samples that have been correctly identified by Sanger sequencing.

[0110] Other parts not described in detail are prior arts. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer set for RT-qPCR detection of multi-host delta coronavirus nucleic acid, characterized in that: The detection primer set includes a pair of primers DCoV-NF / R and a probe; wherein the primer pair RT-F / R is as follows: DCoV-NF: 5'-CAGGTKCTYAARGCTCA-3', DCoV-NR: 5'-AGGMAWAGGATGGAGGAA-3'; The probe sequence is: 5'-CCATGGCTAYTGGCTVCGTTWCA-3'.

2. The RT-qPCR detection primer set according to claim 1, characterized in that: The 5' end of the probe is labeled with a HEX group, and the 3' end is labeled with a BHQ1 group.

3. Use of the RT-qPCR detection primer set according to claim 1 or 2 in preparing an RT-qPCR detection kit for multi-host delta coronavirus.

4. A RT-qPCR detection kit for multi-host delta coronavirus, characterized in that: The kit includes an RT-qPCR detection primer set.

5. The RT-qPCR detection kit according to claim 4, characterized in that: The RT-qPCR detection kit also includes 2×PerfectStartTM Probe One-step qPCR SuperMix, TransScript Probe One-stepRT / RI Enzyme Mix and RNase-free Water.

6. Use of the RT-qPCR detection kit according to claim 4 or 5 in detecting delta coronavirus nucleic acid.

7. A method for detecting nucleic acid of multi-host delta coronavirus, characterized in that: The following steps are involved: 1) Extract DNA from the sample to be tested and then reverse transcribe RNA; 2) using the RNA as a template, performing RT-qPCR using the kit described in claim 4 or 5 to obtain a Ct value; 3) Compare and interpret the Ct value based on the standard curve in the kit: When the Ct value is less than 37, the D-coronavirus nucleic acid test of the sample to be tested is positive; When the Ct value is ≥37, the D-coronavirus nucleic acid test of the sample to be tested is negative.

8. The method according to claim 7, characterized in that: In the step 3), The reaction system of RT-qPCR was a 10 μL reaction system, including 2×PerfectStartTM Probe One-step qPCRSuperMix, TransScript Probe One-step RT / RI Enzyme Mix, upstream primer, downstream primer, probe, template, and RNase-free Water; The final concentrations of the upstream primer and the downstream primer in the reaction system are both 0.5 μmol / L, and the final concentration of the probe in the reaction system is 0.2 μmol / L.

9. The method according to claim 7, characterized in that: In the step 3), the reaction conditions of RT-qPCR are 45°C / 5 minutes, 94°C / 30 seconds, and then 45 cycles, each cycle is 94°C / 5 seconds, 56°C / 30 seconds.

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