Real-time fluorescence RT-PCR detection method for Hobi-like virus

Abstract

The invention belongs to the technical field of biology. The invention provides a primer, a kit and a detection method for real-time fluorescence RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection of a Hobi-like virus. The sequence of the primer is as follows: an upstream primer F: GGTAGTCGTCAATGGTTC, a downstream primer F: GGTAGTCGTCAATGGTTC; the downstream primer R is CCGCTTGAGTTAAGATTC, and the downstream primer R is And the probe primer is FAM (Fatty Amplification Molecule)-CTCGTCACATGCATCTG-BHQ1. The probe primer is The method has excellent specificity, sensitivity and repeatability for detection of the Hobi-like virus, the lowest detection limit is 1 copies / reaction, and the method has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a real-time fluorescent RT-PCR detection method for Hobi-like viruses. Background Art

[0002] Hobi-like virus belongs to the genus Pestivirus of the family Flaviviridae. It is a single-stranded positive-strand RNA virus and a representative new species of atypical pestivirus. It is 12.3kb in size, has one open reading frame, and is flanked by non-coding regions (UTR). The main clinical symptom of HoBi-like virus infection is diarrhea, which is extremely similar to and indistinguishable from the symptoms of bovine viral diarrhea virus infection. Since pestivirus is not a strictly host-specific virus, it can cross-infect cattle, sheep, pigs, goats and other wild ruminants.

[0003] Existing detection methods for HoBi-like viruses can be divided into virus isolation. HoBi-like viruses can be isolated by using bovine kidney cells with reference to the BVDV isolation method; or by using the ACE kit for BVDV, HoBi-like viruses in serum can be detected, but they cannot be effectively distinguished from BVDV; and real-time fluorescence RT-PCR technology is a technology that can detect viruses in real time, quickly, sensitively and accurately, and has been widely used in disease diagnosis.

[0004] However, the existing real-time fluorescence RT-PCR technology also has defects such as poor detection limit. Specifically, the detection limit of the scheme provided in "ATaqMan real-time RT-PCR assay for selective detection ofatypical bovine pestiviruses in clinical samples and biological products" is only 200 copies / reaction; the detection limit of the scheme provided in "Development of a TaqMan assay for sensitive detectionof all pestiviruses infecting cattle, including the emerging HoBi-like strains" is only 45.6 copies; the detection limit of the scheme provided in "Establishment of fluorescent quantitative PCR method for important viral pathogens of bovine respiratory diseases" is only 100 copies / reaction; the detection limit of the scheme provided in "Establishment of one-step fluorescent quantitative PCR detection method for HoBi pestivirus" is only 10 3 copies / μL (converted to 2.5×10 4The detection limit of the solution provided by Chinese patent CN103924006A is only 10 copies / μl (converted to 250 copies / reaction); The detection limit of the solution provided by Chinese patent CN 112430686A is only 10 copies / μl (converted to 250 copies / reaction).

[0005] Therefore, there is a need in the art for a method for detecting Hobi-like viruses that has good specificity, repeatability, and a better detection limit. Summary of the invention

[0006] In view of the defects of the prior art, the present invention aims to provide a primer, a kit and a detection method for real-time fluorescent RT-PCR detection of Hobi-like viruses with a lower detection limit and excellent specificity and repeatability. The method needs to achieve a detection limit of 1 copies / reaction and have no cross-reaction with bovine viral diarrhea virus, foot-and-mouth disease virus, peste des petits ruminants virus, porcine reproductive and respiratory syndrome virus, swine fever virus, etc.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A primer for real-time fluorescence RT-PCR detection of Hobi-like virus, the sequence of the primer is:

[0009] Upstream primer F: GGTAGTCGTCAATGGTTC

[0010] Downstream primer R: CCGCTTGAGTTAAGATTC

[0011] Probe primer: FAM-CTCGTCCACATGGCATCTCG-BHQ1.

[0012] A kit for real-time fluorescent RT-PCR detection of Hobi-like viruses, comprising the above-mentioned primers.

[0013] A real-time fluorescent RT-PCR detection method for Hobi-like virus, the method comprising the steps of:

[0014] (1) Take the sample to be tested;

[0015] (2) Prepare the reaction system as follows: 2×One Step RT-PCR Buffer III: 10 μL, RNA: 4 μL, RNase Free dH 2O: 3.6 μL, PrimeScript TaKaRa Ex Taq HS: 0.4 μL, RT Enzyme MixⅡ: 0.4 μL, upstream primer, downstream primer: 0.4 μL each, probe primer: 0.8 μL;

[0016] (3) The reaction was carried out according to the following scheme: reverse transcription at 42°C for 5 min; pre-denaturation at 95°C for 10 s; denaturation at 95°C for 5 s, annealing at 53°C-58°C for 30 s, and cycled 45 times;

[0017] The upstream primer, downstream primer and probe primer are the primers for real-time fluorescence RT-PCR detection of Hobi-like virus mentioned above.

[0018] Preferably, the reaction system is: 20 μL system: 2×One Step RT-PCR Buffer III: 10 μL, RNA: 4 μL, RNase Free dH 2 O: 3.6μL, PrimeScript TaKaRa Ex Taq HS: 0.4μL, RT Enzyme MixⅡ: 0.4μL, upstream primer, downstream primer: 0.4μL each, probe primer: 0.8μL.

[0019] Preferably, when performing the reaction, the protocol is: reverse transcription at 42°C for 5 min; pre-denaturation at 95°C for 10 s; denaturation at 95°C for 5 s, annealing at 53°C for 30 s, and 45 cycles.

[0020] Beneficial effects of the present invention:

[0021] The present invention has excellent specificity, sensitivity and repeatability for the detection of Hobi-like viruses, with a minimum detection limit of 1 copies / reaction, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The real-time fluorescence RT-PCR annealing temperature of the Hobi-like virus of the present invention is optimized for the amplification curve;

[0023] Figure 2 This is a graph showing the specific detection results of real-time fluorescent RT-PCR of the present invention;

[0024] Figure 3 This is a graph showing the sensitivity detection results of the real-time fluorescence RT-PCR of the present invention. DETAILED DESCRIPTION

[0025] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0026] Example 1

[0027] 1 Materials and methods

[0028] 1.1 Main materials and reagents

[0029] Foot-and-mouth disease virus type O-A bivalent inactivated vaccine and peste des petits ruminants live vaccine were purchased from Tiankang Biological Co., Ltd.; porcine reproductive and respiratory syndrome inactivated vaccine and classical swine fever live vaccine were purchased from Wuhan Keqian Biological Co., Ltd.; Mooleyou (bovine viral diarrhea and infectious rhinotracheitis bivalent live vaccine) was purchased from Jinyu Baoling Biological Pharmaceutical Co., Ltd.; one-step fluorescent RT-PCR reaction solution One Step PrimeScript TM RT-PCR Kit (Perfect Real Time) and viral DNA / RNA extraction kit were purchased from TaKaRa (Dalian) Co., Ltd.; plasmid mini-extraction kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; PUC57-5'UTR recombinant plasmid was purchased from Wuhan Jinkairui Bioengineering Co., Ltd.

[0030] 1.2 Main instruments

[0031] Fluorescence PCR instrument (7500Fast, ABI, USA; qTOWER3G, Analytikjena, Jena, Germany); ultra-micro-nucleic acid and protein analyzer (NanoDrop 2000, Thermo Scientific, USA).

[0032] 1.3 Construction and identification of positive plasmids

[0033] According to the 5'UTR gene sequence of the genome of the Hobi-like virus reference strain (KY091656), the corresponding gene sequence was synthesized by Wuhan Jinkairui Bioengineering Co., Ltd. and cloned into the PUC57 vector to construct the standard plasmid PUC57-5'UTR. After sequencing and identification, the concentration was determined using an ultra-micro nucleic acid protein analyzer, and copies were calculated according to the formula.

[0034] Where C is the measured plasmid concentration in ng / μL.

[0035] 1.4 Primer design

[0036] According to the PCR primer design principles, five pairs of primer sets were designed using PrimerPremier 5 software for the Hobi-like virus 5'UTR gene sequence (KY091656) (Table 1).

[0037] Table 1 Primer probe sequences

[0038]

[0039] 1.5 Primer screening and condition optimization for real-time fluorescence RT-PCR reaction

[0040] The prepared positive plasmid DNA was diluted to a certain concentration and used as a template. A 20μL system was used: 2×OneStep RT-PCR BufferⅢ: 10μL, RNA: 4μL, RNase Free dH2O: 3.6μL, PrimeScript TaKaRa ExTaq HS: 0.4μL, RT Enzyme MixⅡ: 0.4μL, primers (upstream and downstream): 0.4μL each, probe: 0.8μL. Reaction program: 42℃ reverse transcription for 5min; 95℃ pre-denaturation for 10s; [95℃ denaturation for 5s, set gradient temperature between 53℃ and 58℃ (variable 1℃) annealing for 30s (fluorescence collection)] × 45 cycles (using qTOWER3G fluorescence PCR instrument). The designed 5 pairs of primer sets were screened and their optimal annealing temperatures were optimized.

[0041] 1.6 Specificity test

[0042] The purchased vaccines were used to extract RNA using a viral nucleic acid extraction kit, and the extracted RNA (foot-and-mouth disease virus, bovine viral diarrhea virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and peste des petits ruminants virus) was used as a template. 2 O was the negative control, and the optimized real-time fluorescence RT-PCR method was used for amplification to evaluate the specificity of the detection method.

[0043] 1.7 Sensitivity test

[0044] The constructed standard plasmid PUC57-5'UTR was diluted 10 times in series and used as a template. 2 O was the negative control, and the optimized real-time fluorescence RT-PCR method was used for amplification to evaluate the sensitivity of the detection method.

[0045] 1.8 Repeatability test

[0046] The established method was used to perform 10-fold serial dilutions of the standard plasmid PUC57-5'UTR (10 4 , 10 5 , 106 A total of 3 serial dilutions were performed for fluorescence PCR detection. Three replicates were set for each gradient as intra-group replicates, and ddH 2 O was used as the negative control group. The experiment was repeated 3 times at different time periods to evaluate the repetition between groups. The average Ct value, standard deviation and coefficient of variation (CV) were calculated to evaluate the repeatability and stability of the established method.

[0047] 1.9 Sample testing

[0048] The established real-time fluorescence RT-PCR method was used to detect 50 samples of sheep feces collected and 150 samples of imported Dorper sheep sera retained in the laboratory to evaluate whether this method can be used for clinical detection.

[0049] 2 Results

[0050] 2.1 Determination of positive plasmid DNA concentration and conversion of copies

[0051] The plasmids with positive sequencing results were selected and extracted with a plasmid mini-extraction kit. The mass concentration of the recombinant plasmid was measured by an ultra-micro nucleic acid protein analyzer to be 100 ng / μL, which was converted to 3×10 10 copies / μL.

[0052] 2.2 Real-time fluorescence RT-PCR reaction conditions optimization results

[0053] Through comparative experiments, it was found that the five different primer groups showed great differences in amplification results and amplification efficiency. Primer groups PP3 and PP4 were completely unable to amplify; primer groups PP2 and PP5 showed more nonspecific amplification during amplification, with the presence of primer hairpin structures or dimers; primer PP1 had a relatively high amplification efficiency, no primer hairpin structures or dimers, good temperature adaptability, and because the amplified product was shorter, the amplification efficiency was higher and the specific effect was obvious.

[0054] For primer set PP1, fluorescence quantitative PCR was performed at 6 gradient annealing temperatures ranging from 53 to 58°C. The results showed that the higher the temperature, the lower the amplification efficiency ( Figure 1 ), the amplification efficiency is highest when the annealing temperature is 53°C.

[0055] The final reaction conditions were determined to be a 20 μL system: 2×One Step RT-PCR Buffer III: 10 μL, RNA: 4 μL, RNase Free dH 2O: 3.6μL, PrimeScript TaKaRa Ex Taq HS: 0.4μL, RT Enzyme MixⅡ: 0.4μL, Primers (upstream and downstream): 0.4μL each, Probe: 0.8μL. Reaction procedure: 42℃ reverse transcription for 5min; 95℃ pre-denaturation for 10s; [95℃ denaturation for 5s, 53℃ annealing for 30s (fluorescence collection)] × 45 cycles.

[0056] 2.3 Specificity test results

[0057] The optimized real-time fluorescence RT-PCR method was used to extract RNA (foot-and-mouth disease virus, bovine viral diarrhea virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, peste des petits ruminants virus, bovine viral diarrhea virus). The results showed that this method only obtained a specific amplification curve for the Hobi-like virus positive standard plasmid template ( Figure 2 ), while other viral nucleic acids and negative controls showed no amplification curves, indicating that this method has good specificity.

[0058] 2.4 Sensitivity test results

[0059] The recombinant plasmid was diluted 10-fold to a concentration range of 1×10 10 ~1×10 0 The standard solution of copies / reaction was tested using the optimized fluorescence PCR method. The results showed that ( Figure 3 ), the minimum detection limit of the optimized detection method for the standard plasmid PUC57-5'UTR was 1 copy / reaction (converted to 0.05 copies / μl), as shown in Table 2, indicating that the fluorescent PCR method established in this study has extremely high sensitivity.

[0060] Table 2 Ct values ​​for each dilution

[0061]

[0062]

[0063] 2.5 Repeatability test results

[0064] The experimental results show (Table 3), each concentration of standard (10 4 ~10 6 The standard deviation of intra-group detection was between 0.05 and 0.36, and the coefficient of variation was between 0.16% and 1.59%. The standard deviation of inter-group detection was between 0.05 and 0.36, and the coefficient of variation was between 0.21% and 1.36%, indicating that the established method has good stability and repeatability.

[0065] Table 3 Ct values ​​for each dilution

[0066]

[0067] 2.6 Sample testing

[0068] The established real-time fluorescence RT-PCR method was used to detect 50 samples of sheep feces collected and 150 samples of imported Dorper sheep sera retained in the laboratory. No HoBi-like virus was detected in any of the 200 samples.

Claims

1. A primer for real-time fluorescence RT-PCR detection of Hobi-like virus, characterized in that: The sequences of the primers are: Upstream primer F: GGTAGTCGTCAATGGTTC Downstream primer R: CCGCTTGAGTTAAGATTC Probe primer: FAM-CTCGTCCACATGGCATCTCG-BHQ1.

2. A kit for real-time fluorescence RT-PCR detection of Hobi-like virus, characterized in that: The kit comprises the primers according to claim 1.

3. A real-time fluorescence RT-PCR detection method for Hobi-like virus, characterized in that: The method comprises the steps of: (1) Take the sample to be tested; (2) Prepare the reaction system according to the following scheme: 2×One Step RT-PCR Buffer III: 10 μL, RNA: 4 μL, RNaseFree dH2O: 3.6 μL, PrimeScript TaKaRa Ex Taq HS: 0.4 μL, RT Enzyme Mix II: 0.4 μL, upstream primer, downstream primer: 0.4 μL each, probe primer: 0.8 μL; (3) The reaction was carried out according to the following scheme: reverse transcription at 42°C for 5 min; pre-denaturation at 95°C for 10 s; denaturation at 95°C for 5 s, annealing at 53°C-58°C for 30 s, and cycled 45 times; The upstream primer, downstream primer and probe primer are the primers for real-time fluorescent RT-PCR detection of Hobi-like virus as claimed in claim 1.

4. The detection method according to claim 3, characterized in that: The reaction system is: 20μL system: 2×One StepRT-PCR BufferⅢ: 10μL, RNA: 4μL, RNase Free dH2O: 3.6μL, PrimeScript TaKaRa Ex TaqHS: 0.4μL, RT Enzyme MixⅡ: 0.4μL, upstream primer, downstream primer: 0.4μL each, probe primer: 0.8μL.

5. The detection scheme according to claim 3, characterized in that: The reaction protocol was as follows: reverse transcription at 42°C for 5 min; pre-denaturation at 95°C for 10 s; denaturation at 95°C for 5 s, annealing at 53°C for 30 s, and 45 cycles.

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