A kind of primer, probe and application of TaqMan fluorescent quantitative RT-PCR detection of bovine newb virus

Abstract

The present invention discloses a bovine newb virus TaqMan fluorescent quantitative RT-PCR detection primer, probe and application. It belongs to the field of biological detection technology. It includes: primers NB-F-4716, NB-R-4949 and probe NBV-TZ. The optimal concentrations of upstream and downstream primers of the present invention are both 500nmol / L, the probe concentration is 400nmol / L, and the concentration of the primer is 1×10 8 ~1×10 1 There is a good linear relationship between copies / μL, and the linear correlation coefficient R 2 =0.996, the amplification efficiency was 105.9%; the specificity was strong, the sensitivity was high, and the minimum detection limit for the BNeV plasmid standard was 1×10 1 The repeatability was good, and the coefficient of variation within and between groups was less than 3%, which provided a powerful means for the detection of BNeV and molecular epidemiological investigation.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and more specifically to a bovine new bronchitis virus TaqMan fluorescent quantitative RT-PCR detection primer, a probe and an application thereof. Background Art

[0002] Nebovirus (NeV) is the only member of the Caliciviridae family and the Nebovirus genus. Other viruses in the same family include the Norovirus, Sapovirus, Vesivirus, and Lagovirus genera.

[0003] NeV is a non-enveloped single-stranded positive-strand RNA virus with a genome length of about 7.4 kb, consisting of two open reading frames (ORFs), and divided into three genotypes: NB, NA1 and DijonA216. The only susceptible animal to the Newb virus is cattle, which can induce anorexia and xylose malabsorption in calves. Although there is no isolation and culture system for NeV, it can cause severe diarrhea and intestinal damage to newborn sterile calves after aseptic treatment of diarrheal feces containing NeV that excludes other viruses, especially in the duodenum and jejunum.

[0004] BNeV has been detected in calf feces samples in the United States, the United Kingdom, Brazil and my country. Its molecular biological detection methods include SYBR Green fluorescence quantitative RT-PCR detection method, TB Green fluorescence quantitative RT-PCR detection method, nested RT-PCR detection method and conventional RT-PCR detection method. There have been no reports on TaqMan fluorescence quantitative RT-PCR detection of bovine Newbovirus.

[0005] Therefore, how to provide a bovine new bronchitis virus TaqMan fluorescent quantitative RT-PCR detection primer and a detection method and system to provide more accurate results is a problem that technical personnel in this field urgently need to solve. Summary of the invention

[0006] In view of this, the present invention provides a bovine new bronchitis virus TaqMan fluorescent quantitative RT-PCR detection primer, a probe and an application thereof.

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

[0008] A TaqMan fluorescent quantitative RT-PCR detection primer for bovine new bronchitis virus comprises: a primer NB-F-4716, the nucleotide sequence of which is shown in SEQ ID NO.1; and a primer NB-R-4949, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] Preferably, the probe NBV-TZ is also included, and the nucleotide sequence is shown in SEQ ID NO.3.

[0010] The present invention also provides a reagent or a kit containing the primer.

[0011] The present invention also provides a method for detecting bovine new bronchitis virus using TaqMan fluorescent quantitative RT-PCR for non-diagnostic and therapeutic purposes, using the above-mentioned primers.

[0012] The preferred amplification system includes: GoTaq Probe qPCR Master Mix 10μL, ScriptTMRT-MIX for1-Step RT-qPCR 2×0.4μL, 10μmol / L upstream and downstream primers 1μL each, 10μmol / L probe 0.8μL, cDNA template 2μL, and the remainder RNase-Free ddH2O, with a total volume of 20μL.

[0013] Preferably, the amplification procedure includes: reverse transcription at 45°C for 15 min; pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for a total of 45 cycles.

[0014] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a bovine Newb virus TaqMan fluorescent quantitative RT-PCR detection primer, probe and application, and the technical effect achieved is that the minimum detection amount of BNeV fluorescent quantitative RT-PCR method established by the present invention is 1.0×10 1 copies / μL, at 1.0×10 6 ~1.0×10 2 The 5 gradient ranges of 100 copies / μL showed good repeatability (coefficient of variation <3.0%).

[0015] In addition, because the nucleic acid in fecal samples is easily degraded, a nucleic acid protective agent is added when collecting fecal samples, which are placed in a dry freezer and quickly transported to a -80°C refrigerator for storage, minimizing RNA degradation and effectively increasing the detection rate of viral nucleic acid.

[0016] The variability of the Newb virus gene sequence was taken into consideration when designing the primers, and the sensitivity of the present invention was improved by using degenerate primer amplification. After optimizing the primers and probes, the present invention established a TaqMan fluorescent quantitative RT-PCR method with strong specificity, high sensitivity and good stability. The sensitivity was higher than the nested RT-PCR method and the common RT-PCR detection method reported in the literature, and the sensitivity was 100 times higher than that of the common RT-PCR. At the same time, the fecal viral load was detected, and the average viral load of diarrhea fecal samples was 8.7×10 5Only one positive stool sample was detected, with a viral load of 2.6×10 3 The BNeV positive rate of the sample was 35.1%. The present invention analyzes the viral load in calf feces samples for the first time, providing effective technical support for the early diagnosis and molecular epidemiological investigation of BNeV. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0018] Figure 1 The attached drawing is a diagram of the optimization results of the BNeV fluorescent quantitative RT-PCR primers provided by the present invention, wherein A-500nmol / L; B-400nmol / L; C-300nmol / L; D-200nmol / L; E-100nmol / L.

[0019] Figure 2 The attached drawing is a diagram of the optimization results of the BNeV fluorescent quantitative RT-PCR probe provided by the present invention, wherein A-500nmol / L; B-400nmol / L; C-300nmol / L; D-200nmol / L; E-100nmol / L.

[0020] Figure 3 The accompanying drawing is a standard curve diagram of BNeV fluorescence quantitative RT-PCR provided by the present invention.

[0021] Figure 4 The attached drawing is a diagram showing the results of a BNeV fluorescent quantitative RT-PCR specificity test provided by the present invention, wherein A.-Bovine Newbub virus; B-other pathogens and negative controls.

[0022] Figure 5 The attached figure is a diagram showing the results of the BNeV fluorescence quantitative RT-PCR sensitivity test provided by the present invention, wherein A to H: 1.0×10 8 ~1.0×10 1 copies / μL plasmid DNA; I. Negative control and 1.0×10 0 ·copies / μL plasmid DNA.

[0023] Figure 6The attached figure is a diagram showing the results of the BNeV common RT-PCR sensitivity test provided by the present invention, wherein M.500bpLadder DNA Marker A~I: 1.0×10 8 ~1.0×10 0 copies / μL plasmid DNA; J. Negative control.

[0024] Figure 7 The attached figure is a graph showing the results of the bovine new bronchitis virus fluorescence quantitative RT-PCR repeatability test provided by the present invention, wherein A to E: 1.0×10 6 ~1.0×10 2 copies / μL plasmid DNA; F. Negative control. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The embodiment of the invention discloses a bovine new brucella virus TaqMan fluorescent quantitative RT-PCR detection primer, a probe and an application thereof.

[0027] In the examples, the experimental methods, raw materials and equipment not mentioned are all conventional experimental methods and commercially available experimental materials, which will not be described in detail here.

[0028] Among them, viruses, bacteria and clinical samples:

[0029] Bovine viral diarrhea virus types 1 and 2, bovine coronavirus, bovine rotavirus, Escherichia coli, and Salmonella were all preserved by the Department of Infectious Diseases, College of Veterinary Medicine, Inner Mongolia Agricultural University; 37 calf feces samples were obtained from ranches in Inner Mongolia, and all samples were stored at -80°C.

[0030] Main reagents:

[0031] RNeasy Mini Kit was purchased from QIAGEN; GoTaq Probe qPCR and RT-qPCR Systems and One Step qRT-PCR Kit were purchased from Promega; pMD19-T vector, DH5ɑ competent cells, PremixTaqTM, EASY Dilution, 6×Loading Buffer, and 500bp DNA LadderMarker were purchased from TaKaRa; DNA gel recovery kit was purchased from Axygen; plasmid mini-extraction kit and RNase-Free ddH2O were purchased from TIANGEN.

[0032] Design and synthesis of primers and probes:

[0033] The primers were synthesized by Shanghai Bioengineering Co., Ltd., and the probes were synthesized by Takara Biotechnology (Beijing) Co., Ltd.

[0034] Example 1

[0035] According to the BNebV-RdRp gene sequence of GenBank (MH718886, MG599036, MW036464), the MegAlign program in Lasergener software was used for homology analysis, and a pair of BNeV primers and probes were designed by selecting conservative sequences. The sequences are shown in Table 1.

[0036] Table 1 Primer and probe sequence information for amplifying BNV

[0037]

[0038]

[0039] Example 2

[0040] Preparation of standards

[0041] The sample RNA was extracted according to the instructions of the RNeasy Mini Kit, and the target gene was amplified using the upstream and downstream primers in Table 1. The PCR product was purified by gel recovery and ligated to the pMD19-T vector (ligation reaction system: pMD19-T Vector 1.0 μL; purified product 4.0 μL; Solution I 5.0 μL; reaction conditions: overnight culture at 4°C) and transformed into Escherichia coli DH5α competent cells, plated, positive clones were screened and cultured (specific steps:

[0042] Conversion:

[0043] (1) When the DH5α competent cells are thawed in ice until they are semi-foggy, add 5 μL of the ligation product to 50 μL of the DH5α competent cells, flick to mix, and place on ice for 30 min.

[0044] (3) Take out the mixed solution and immediately place it in a 42°C metal bath for 45 seconds and then immediately place it in an ice box for 2 minutes.

[0045] (4) Add 890 μL of LB liquid medium without Amp to the mixture, and then place it on a shaker at 37°C and 200 rpm for 1 h.

[0046] (5) Centrifuge at 4000 rpm for 3 min, discard 795 μL of supernatant, and mix the remaining 150 μL of supernatant and bacterial pellet.

[0047] (6) Spread it evenly on LB solid medium containing Amp, invert and culture it in a 37°C incubator overnight, and observe whether single colonies appear.

[0048] Colony expansion culture

[0049] Pick 6 single colonies on the culture medium, add them to 5 mL of LB liquid culture medium with Amp, mix well and culture overnight in a constant temperature shaking incubator at 37°C and 200 rpm, extract the plasmid for sequencing and identification. The positive recombinant plasmid identified correctly is named pMD19T-BNeV, the recombinant plasmid DNA concentration is measured, and the copy number is calculated.

[0050] Example 3

[0051] Optimization of Fluorescence Quantitative RT-PCR Conditions

[0052] The template was the positive recombinant plasmid DNA of bovine newbub virus, and 0.2 μL, 0.4 μL, 0.6 μL, 0.8 μL, and 1 μL of the upstream and downstream primers were respectively aspirated to optimize the primer concentration.

[0053] Then take the optimized primer concentration, and absorb 0.2μL, 0.4μL, 0.6μL, 0.8μL, and 1μL of the probe respectively. The template is the positive recombinant plasmid DNA of bovine new bronchitis virus, and then optimize the probe concentration.

[0054] In the 20μL amplification system, the minimum CT value and the highest fluorescence were selected as the reference standard. The results showed that the optimal upstream and downstream primer concentrations of BNV were both 500nmol / L, and the optimal probe concentration was 400nmol / L. The optimization curve is shown in Figure 1 and Figure 2 .

[0055] Fluorescence quantitative PCR amplification was performed using the optimized primers and probes. The amplification system was as follows: GoTaq ProbeqPCRMaster Mix 10μL, ScriptTMRT-MIX for 1-Step RT-qPCR 2×0.4μL, 10μmol / L upstream and downstream primers 1μL each, 10μmol / L probe 0.8μL, cDNA template 2μL, RNase-Free ddH2O 4.8μL, total volume 20μL; amplification program: reverse transcription at 45℃ for 15min; pre-denaturation at 95℃ for 2min; denaturation at 95℃ for 15s, annealing at 55℃ for 30s, and extension at 72℃ for 30s, for a total of 45 cycles.

[0056] Example 4

[0057] Establishment of standard curve

[0058] The concentration was selected as 1.0×10 8 ~1.0×10 1 The optimized fluorescence quantitative RT-PCR amplification system and procedure were used to amplify the 1000 copies / μL plasmid DNA as template, and enzyme-free water was used as negative control to draw a standard curve.

[0059] Standard curve of fluorescent quantitative RT-PCR

[0060] The concentration was selected as 1.0×10 8 ~1.0×10 1 The 1000 copies / μL plasmid DNA was used as a template for amplification to obtain the amplification curve and standard curve of bovine new bronchitis virus fluorescence quantitative RT-PCR (see Figure 3 ), the results showed that the slope of the standard curve parameter of BNV was -3.189; R 2 The value was 0.996; the amplification efficiency (E) was 105.9%, indicating that the established method had high relevance and could be used for the quantitative detection of BNV.

[0061] Experimental results:

[0062] Specificity test

[0063] The RNA / DNA extraction kit was used to extract the nucleic acids of bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus, Escherichia coli, and Salmonella according to the instructions of the RNA / DNA extraction kit. 6 The positive recombinant plasmid standard of bovine new bronchitis virus with 100 copies / μL was used as the positive control, and enzyme-free water was used as the negative control. The bovine new bronchitis virus fluorescent quantitative RT-PCR method established in the example was used for detection to evaluate the specificity of the method.

[0064] The results show that:

[0065] The bovine Newbub virus fluorescent quantitative RT-PCR method established in the example was used to detect bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus, Escherichia coli and Salmonella nucleic acids. The results showed that only bovine Newbub virus was detected, and no amplification curves were found for other pathogens and negative controls, which proved that the method had good specificity and could be used for bovine Newbub virus detection. Figure 4 .

[0066] Sensitivity test

[0067] The concentration was selected as 1.0×10 8 ~1.0×10 0 The copies / μL plasmid DNA was used as template for conventional RT-PCR and fluorescent quantitative RT-PCR amplification, and the lowest copy number that could be detected by the two detection methods was obtained.

[0068] The results showed that the minimum detection amount of BNV by qPCR was 1.0×10 1 copies / μL, while the minimum detection amount of common RT-PCR is 1.0×10 3 copies / μL, indicating that the method established by the present invention has good sensitivity. Figure 5 , 6 .

[0069] Repeatability test

[0070] The concentration was selected as 1.0×10 6 ~1.0×10 2 The plasmid DNA of 5 gradients of 100 copies / μL was amplified using the bovine new bronchitis virus fluorescent quantitative RT-PCR method established in the example, and the intra-group and inter-group repeatability tests were performed. The coefficient of variation of the intra-group and inter-group Ct values ​​were calculated respectively to detect the repeatability of the method.

[0071] The results showed that: after three repeatability tests, the coefficient of variation of the Ct values ​​within and between groups was less than 3.0%, indicating that the method had good repeatability and stability. Figure 7 .

[0072] Table 2 Results of intra-group and inter-group repeatability tests of bovine new bronchitis virus fluorescence quantitative RT-PCR

[0073]

[0074] Testing of clinical samples

[0075] According to the instructions of the RNA rapid extraction kit, nucleic acids from 37 clinical samples were extracted, and 37 calf feces samples collected from pastures in Inner Mongolia were tested using the bovine new bronchitis virus fluorescent quantitative RT-PCR method and the common RT-PCR method established in the example.

[0076] The conventional RT-PCR method and the fluorescent quantitative RT-PCR method for bovine Newbub virus established by the present invention were used to detect 37 calf feces samples collected, and positive and negative controls were set at the same time. The conventional RT-PCR method only detected 4 bovine Newbub virus-positive diseased materials, with a positive rate of 10.8%, while the fluorescent quantitative RT-PCR method established by the embodiment detected 13 bovine Newbub virus-positive diseased materials, with a positive rate of 35.1%, indicating that the fluorescent quantitative RT-PCR method established by the present invention is more sensitive than the conventional RT-PCR method. Table 3 is the detection data of bovine Newbub virus virus load in calf feces samples.

[0077] Table 3 Detection of BNV viral load in calf feces

[0078]

[0079] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0080] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein. Sequence Listing <110> Inner Mongolia Agricultural University <120> A kind of primer, probe and application of TaqMan fluorescent quantitative RT-PCR detection of bovine newb virus <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 gcgctctgct kgacctaac 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <400> 2 cgcgggcttc ttctagaga 19 <210> 3 <211> twenty three <212> DNA <213> Artificial Sequence <400> 3 cctcggcgtt ccgtgtggga cca 23

Claims

1. A TaqMan fluorescent quantitative RT-PCR detection primer and probe for bovine new bronchitis virus, characterized in that: include: Primer NB-F-4716, the nucleotide sequence is shown in SEQ ID NO.1; The primer NB-R-4949 has a nucleotide sequence as shown in SEQ ID NO.2; and the probe NBV-TZ has a nucleotide sequence as shown in SEQ ID NO.

3.

2. A reagent or kit comprising the primers and probes according to claim 1.

3. A method for detecting bovine new bovine virus using TaqMan fluorescent quantitative RT-PCR for non-diagnostic and therapeutic purposes, characterized in that: The primers and probes according to claim 1 are used.

4. The method according to claim 3, characterized in that The amplification system includes: GoTaq Probe qPCRMasterMix 10 µL, ScriptTMRT-MIX for 1-Step RT-qPCR 2× 0.4 µL, 1 µL each of 10 µmol / L upstream and downstream primers, 0.8 µL of 10 µmol / L probe, 2 µL of cDNA template, and the remaining amount of RNase-Free ddH2O, with a total volume of 20 µL.

5. The method according to claim 4, characterized in that The amplification procedure included: reverse transcription at 45°C for 15 min; pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for a total of 45 cycles.

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