Quadruple fluorescent quantitative PCR (Polymerase Chain Reaction) primer probe group for rapidly detecting four pathogens of birds and application of quadruple fluorescent quantitative PCR primer probe group

Abstract

The invention discloses a quadruple fluorescent quantitative PCR (Polymerase Chain Reaction) primer probe group for rapidly detecting four pathogens of birds and application, and belongs to the technical field of biology. The kit comprises specific primers and probes aiming at coronavirus (gamma, delta), avian influenza virus (H9 subtype), Newcastle disease virus and West Nile virus; the reagent disclosed by the invention is used for detecting four viruses carried by birds, and has the advantages of high specificity, strong sensitivity, short detection time, safety, low cost and the like; the method can realize detection of pathogens carried by birds, and is of great significance to customs frontier defense and quarantine work.

Description

Technical Field

[0001] The invention relates to a quadruple fluorescent quantitative PCR primer probe set for rapidly detecting four bird pathogens and application thereof, belonging to the field of biotechnology. Background Art

[0002] The habitat and migration behavior of wild birds and their unique adaptive immune systems provide a natural reservoir for many virus species, and also provide more possibilities for the outbreak and spread of pathogenic viruses. Human invasion of the natural habitats of wild birds, the domestication of wild birds, and the increase in human consumption of poultry have resulted in a large overlap of activities in human habitats, leading to increased interactions between wild animals and domestic poultry, as well as within animal groups. Transmission between migratory birds and poultry, and indirectly to humans, provides opportunities for cross-species transmission of avian viruses, and mutual transmission between wild birds and domestic poultry, which deserves attention in poultry.

[0003] Currently, the detection technologies available on the market mainly include methods for detecting duck-origin viruses, pigeon-origin viruses, chicken-origin viruses and bacteria, but it is not common to detect important pathogens carried by wild birds. However, infectious pathogens carried by birds are often reported. For example, the H7N9 subtype avian influenza that broke out in 2013, the virus gene originated from the gene reassortment between wild birds and poultry in East Asia, and the H9N2 subtype is widely present in my country and is the main subtype of avian influenza currently prevalent; there are more than 16,000 cases of West Nile virus infection in the Americas; in 1926, Newcastle disease virus was reported in more than 100 countries in South America, Asia, Europe and Africa, and the disease has a high morbidity and mortality rate.

[0004] The present invention determines four pathogens according to the pathogen spectrum carried by wild birds, namely: avian influenza virus H9 subtype (avian influenza virus), coronavirus (γ, δ genus) (coronavirus), Newcastle disease virus (Newcastle disease virus), West Nile virus (West Nile Virus), wherein avian influenza virus belongs to influenza A virus, which is very easy to undergo recombination and mutation to produce multiple subtypes, mainly causing respiratory system lesions, and highly pathogenic subtypes may even cause systemic sepsis in the host, and may infect a variety of birds and mammals. Coronavirus is commonly found in wild birds around the world, and is the natural host of γ coronavirus and δ coronavirus. Newcastle disease caused by Newcastle disease virus is one of the most destructive diseases, with extremely fast transmission speed, and strong strains can destroy the entire flock of chickens, seriously weakening the global poultry industry; its genome is a non-segmented, negative-sense single-stranded RNA. Infected humans mainly have symptoms such as conjunctivitis or lymphadenitis; West Nile Virus (WNV) is a member of the Flaviviridae family. Human infection is associated with febrile illness, which can progress to fatal encephalitis, leading to cognitive dysfunction and flaccid paralysis.

[0005] Therefore, the invention establishes a reagent capable of rapidly detecting pathogenic viruses carried by wild birds, which is of great significance for preventing and controlling diseases spread by wild birds. Multiplex fluorescence quantitative PCR technology has the advantages of strong specificity, high sensitivity, simple operation, short time and low cost in detecting major pathogens carried by wild birds. Summary of the invention

[0006] In response to the need for quarantine of avian epidemic diseases, one of the objectives of the present invention is to provide a quadruple fluorescent quantitative PCR primer probe set for rapid detection of four avian pathogens.

[0007] The quadruple fluorescent quantitative PCR primer probe includes specific primers and probes for coronavirus, Newcastle disease virus, avian influenza virus H9, and West Nile virus, wherein the upstream primer of coronavirus is shown in SEQ ID NO:1, the downstream primer is shown in SEQ ID NO:2, and the probe is shown in SEQ ID NO:3; the upstream primer of Newcastle disease virus is shown in SEQ ID NO:4, the downstream primer is shown in SEQ ID NO:5, and the probe is shown in SEQ ID NO:6; the upstream primer of avian influenza virus H9 is shown in SEQ ID NO:7, the downstream primer is shown in SEQ ID NO:8, and the probe is shown in SEQ ID NO:9; the upstream primer of West Nile virus is shown in SEQ ID NO:10, the downstream primer is shown in SEQ ID NO:11, and the probe is shown in SEQ ID NO:12.

[0008] Another object of the present invention is to provide a quadruple fluorescent quantitative PCR primer probe set for rapid detection of four bird pathogens, and its use in detecting avian coronavirus, Newcastle disease virus, avian influenza virus H9, and West Nile virus.

[0009] Preferably, the specific steps include:

[0010] (1) Use an RNA extraction kit to extract RNA from throat swabs, anal swabs, stool or tissue samples.

[0011] (2) Using the RNA obtained in step (1) as a template, using the primers and probes shown in SEQ ID NO: 1 to SEQ ID NO: 12 to perform fluorescent quantitative PCR detection, and determining the results based on the Ct value.

[0012] Preferably, the reaction system for fluorescence quantitative PCR detection in step (2) is 20 μL of 2×Pro TaqHS Probe Premix II, 4 μL of template, 1 uL of upstream and downstream primers of avian influenza virus H9, 1.2 uL of upstream and downstream primers of Newcastle disease virus and West Nile virus, 0.5 uL of probe of avian influenza virus H9, 0.6 uL of probe of coronavirus, Newcastle disease virus and West Nile virus, and sterile water is added to make up to 40 μL.

[0013] Preferably, the reaction procedure of the fluorescence quantitative PCR detection in step (2) is reverse transcription at 42°C for 5 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing and extension at 56°C for 30 s, 40 cycles; and collecting the fluorescence signal during the annealing and extension stage of each cycle.

[0014] The interpretation of the test results includes the following: (1) If the amplification curve shows a standard "S" shape without abnormal fluctuations, and there is no Ct value in the negative control group and the no-template control group, the experiment is considered valid. If it does not meet the requirements, multiplex fluorescence quantitative PCR detection must be performed again, or nucleic acid must be re-extracted for quadruple fluorescence quantitative PCR detection; (2) If the pathogen Ct value is ≤36.0, it is positive. If 36<Ct≤40, and the re-test is still 36<Ct≤40, it is negative; (3) If the CT value shows UNDFT, the sample is below the detection limit and the result is negative.

[0015] Beneficial Effects of the Invention

[0016] The present invention is the first to establish a multiplex fluorescence quantitative PCR detection method for four pathogenic pathogens carried by birds. The specificity evaluation of the method found that there was no cross reaction between the pathogens, and the specificity was good. The four viruses could be accurately distinguished. The sensitivity evaluation found that the detection limit of avian influenza virus H9 and West Nile virus could reach 10 2The sensitivity of this method is only on the order of copies / μL, while that of coronavirus and Newcastle disease virus can reach 10 copies / μL. The repeatability evaluation of this method found that the coefficient of variation (CV) of each group of pathogens within and between batches was less than 5, indicating good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the reaction result of the quadruple fluorescence quantitative PCR system in Example 3.

[0018] Figure 2 These are the results of the single-plex qPCR specificity experiment of coronavirus (γ and δ genera) in Example 3.

[0019] Figure 3 These are the results of the single-plex qPCR specificity experiment of avian influenza virus (H9 subtype) in Example 3.

[0020] Figure 4 These are the results of the West Nile virus single-plex qPCR specificity experiment in Example 3.

[0021] Figure 5 This is the specificity experimental result of Newcastle disease virus single qPCR in Example 3.

[0022] Figure 6 These are the results of the single-plex qPCR sensitivity experiment of coronavirus (γ and δ genera) in Example 3.

[0023] Figure 7 The results of the single-plex qPCR sensitivity experiment of avian influenza virus (H9 subtype) in Example 3.

[0024] Figure 8 This is the result of the West Nile virus single-plex qPCR sensitivity experiment in Example 3.

[0025] Fig. 9 This is the result of the Newcastle disease virus single-plex qPCR sensitivity experiment in Example 3. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following embodiments are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0027] Example 1

[0028] Design of specific primers and probes

[0029] (1) The pathogen gene reference sequences were downloaded from the NCBI (National Center for Biotechnology Information) website as follows: 72 sequences of HA gene of avian influenza virus, Rdrp region of coronavirus, F gene of Newcastle disease virus, and E gene of West Nile virus;

[0030] (2) Nucleotide sequences were aligned using Mega7 software, and primers and probes were designed using Primer Select software;

[0031] (3) Primer and probe BLAST evaluation: The nucleotide sequences of the primers and probes that have been preliminarily designed are compared again using the BLAST search function on the NCBI website to select primers and probe sequences with high specificity;

[0032] The nucleotide sequences of specific primers and probes targeting pathogens carried by four species of wild birds are shown in Table 1 (SEQ ID NO: 1-SEQ ID NO: 12).

[0033] Table 1 Nucleotide sequences of specific primers and probes for pathogens carried by 14 bird species

[0034]

[0035] Example 2

[0036] Establishment of quadruple fluorescence quantitative PCR method

[0037] 1. Construction of plasmid

[0038] The specific gene sequences of AIV, COV, NDV, and WNV were connected to the pBluescript II SK(+) vector. The specific gene sequences are shown in SEQ ID NO: 13 to SEQ ID NO: 16. Four virus-positive reference plasmids were synthesized. The plasmid construction was completed by Bioengineering (Shanghai) Co., Ltd. The concentration was measured by an ultraviolet spectrophotometer, and the copy number of the plasmid was calculated according to the length and concentration of each virus-positive reference plasmid. The calculation formula is as follows, and the calculation results are shown in Table 2:

[0039]

[0040] Table 2 Fragment size, plasmid concentration and copy number of each virus-positive reference plasmid

[0041]

[0042] 2. Optimization of multiple fluorescence quantitative reaction system

[0043] The copy number is 10 4Four virus-positive reference plasmids with 10 copies / μL were used as templates, and the Pro Taq HS premixed probe kit from Acori Biotech was used for quadruple fluorescence quantitative PCR detection. The real-time fluorescence quantitative PCR instrument was Biori FQD-96a.

[0044] The reaction system consisted of 2×Pro TaqHS Probe PremixⅡ20μL, template 4μL, upstream primer, downstream primer, and probe, and sterile water was added to make up to 40μL. In order to obtain the optimal amount of primers and probes added, 9 groups of different primers and probes were explored. The specific amounts of the 9 groups of primers and probes are shown in Table 3, and the Tm values ​​were 56, 58, and 60℃, respectively. The reaction procedure was: reverse transcription at 42℃ for 5min, pre-denaturation at 95℃ for 30s; denaturation at 95℃ for 5s, annealing and extension at 56℃ for 30s, and 40 cycles. The fluorescence signal was collected during the annealing and extension stage of each cycle.

[0045] Table 3 9 different primer and probe dosages investigated

[0046]

[0047] By comparing the average value and standard deviation of the Ct value, as well as comprehensive indexes such as the amplification curve of the test group, it was determined that the dosage of upstream and downstream primers for avian influenza virus (H9 subtype), coronavirus, Newcastle disease virus, and West Nile virus was 1.2uL, the dosage of probes for avian influenza virus H9 was 0.5uL, and the dosage of probes for coronavirus, Newcastle disease virus, and West Nile virus was 0.6uL. When the Tm value was 56°C, the reaction system was optimal.

[0048] Example 3

[0049] Evaluation of specificity, sensitivity and repeatability of fluorescent quantitative PCR detection primers of the present invention

[0050] 1. Evaluation of specificity of fluorescent quantitative PCR

[0051] (1) Quadruple fluorescence quantitative PCR detection of mixed samples

[0052] Select 10 4 Four virus-positive reference plasmids with 100 copies / μL and four other pathogens carried by birds: circovirus, avian astrovirus, hepatitis B virus, and rotavirus were used as templates for quadruple fluorescent quantitative PCR detection, among which avian astrovirus and rotavirus were first reversed into cDNA, and the negative control was ddH2. Quadruple fluorescent quantitative PCR detection was performed, and the reaction system was shown in Table 4. In the quadruple fluorescent quantitative PCR detection, the template was a single pathogen or virus-positive reference plasmid, and four primers and probes were added at the same time.

[0053] Table 4 Quadruple fluorescence quantitative PCR detection system

[0054]

[0055] The amplification program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing and extension at 56°C for 30 s, 40 cycles, and the fluorescence signal was collected during the annealing and extension phase of each cycle;

[0056] The results are as follows Figure 1 As shown; only the virus-positive reference plasmid showed an amplification curve, while the other four viruses had no amplification curve, indicating that the quadruple fluorescence quantitative PCR detection had good specificity.

[0057] (2) Evaluation of single-plex fluorescence quantitative PCR specificity

[0058] Then, specific primers and probes of coronavirus, avian influenza virus (H9 subtype), Newcastle disease virus, and West Nile virus were used to detect four virus-positive reference plasmid mixed templates (the concentration of virus-positive reference plasmids in the mixed template was the same). When single-plex fluorescence quantitative PCR was performed, only one primer and probe set was added to each reaction. The amount of each primer and probe added is shown in Table 5. The amplification program was: pre-denaturation at 95°C for 30s; denaturation at 95°C for 5s, annealing and extension at 56°C for 30s, 40 cycles, and the fluorescence signal was collected during the annealing and extension stage of each cycle; the results are shown in Table 5. Figure 2-5 , Figure 2 The results of amplification using coronavirus primers and probe sets are shown in Figure 2. Figure 3 The results of amplification using the avian influenza virus H9 primer and probe set are shown in Figure 2. Figure 4 The results of amplification using the West Nile virus primer and probe set are shown in Figure 2. Figure 5 This is the result of amplification using Newcastle disease virus and probe set primers. From the above results, it can be seen that there is no cross-reaction between the pathogens in each group, indicating that the single-plex fluorescent quantitative PCR has good specificity.

[0059] Table 5 Single-plex fluorescence quantitative PCR detection system

[0060]

[0061] 2. Evaluation of fluorescence quantitative PCR sensitivity

[0062] Using the four virus positive reference plasmids as templates, the prepared four virus positive reference plasmids were diluted to 10 by 10-fold dilution method. 7 copies / μL, 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 102 copies / μL, 10 1 copies / μL, and specific primers and probes for the four viruses carried by birds were used to detect the minimum detection concentration of each virus. The reaction system is shown in Table 4. Figure 6-9 As shown, the amplification curves from left to right are 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 (copies / uL), the results show that the detection system of the present invention can detect viruses carried by four wild birds with a minimum detection limit of 10 for avian influenza virus H9 and West Nile virus. 2 copies / μL, while the sensitivity of coronavirus and Newcastle disease virus can reach 10 1 copies / μL order of magnitude.

[0063] 3. Evaluation of the repeatability of quadruple fluorescence quantitative PCR

[0064] In order to verify the repeatability of the quadruple qPCR assay, the concentration of 5 -10 3 Four virus-positive reference plasmids with 4 copies / μL were used as templates for quadruple fluorescence quantitative PCR experiments. Biori FQD-96a real-time fluorescence quantitative PCR instrument was used for detection. The reaction system and amplification program were the same as those in Table 4. Fluorescence signals were collected during the annealing and extension phase of each cycle.

[0065] Conduct intra-group and inter-group repeatability experiments respectively; use specific primers and probes of viruses carried by four wild birds to detect each plasmid template; intra-batch repeatability: repeat three times at the same time, observe and record the Ct value; inter-batch repeatability: repeatability detection is performed every Wednesday for three consecutive weeks, and the Ct value is observed and recorded. Calculate the CV value according to the following formula:

[0066]

[0067] The coefficient of variation (CV) of each virus was less than 5 when the template concentration was the same, indicating that the qPCR method had good repeatability. The repeatability results are shown in Table 6.

[0068] Table 6 Quadruple fluorescence quantitative PCR replicate results

[0069]

[0070] Example 4

[0071] Source and processing of test samples

[0072] 1. Sample source

[0073] Throat swabs, anal swabs and feces of birds collected from various states are collected, marked, and recorded. They are stored at 4°C during transportation and stored at -80°C until nucleic acid extraction.

[0074] 2. Nucleic acid extraction

[0075] RNA was extracted using the Tiangen total RNA extraction kit. The specific steps are as follows:

[0076] a. Before the experiment, add anhydrous ethanol to the rinse solution RW before using the Tiangen Total RNA Extraction Kit. Please refer to the label on the bottle for the amount of ethanol to be added.

[0077] b. Sample pretreatment: After the tissue is divided and added with grinding beads, 1 mL of lysis buffer RZ is added for every 50-100 mg of tissue, and homogenized with a homogenizer; the homogenized sample is placed at room temperature for 5 minutes to completely separate the nucleic acid-protein complex;

[0078] c. Add 200uL chloroform, cover the tube, shake vigorously for 15 seconds, and leave at room temperature for 3 minutes;

[0079] d. Centrifuge at 4℃ 12000rpm (~13400xg) for 10min. The sample will separate into three layers: yellow organic phase, middle layer and colorless aqueous phase. RNA is mainly in the aqueous phase, and the volume of the aqueous phase is about 50% of the lysis solution RZ reagent used. Transfer the aqueous phase to a new tube and proceed to the next step.

[0080] e. Slowly add 0.5 times volume of anhydrous ethanol, mix well (precipitate may appear at this time), transfer the obtained solution and precipitate into adsorption column CR3, centrifuge at 4℃12,000rpm (~13,400xg) for 30sec. If all the solution and mixture cannot be added to adsorption column CR3 at one time, transfer them into adsorption column CR3 twice, centrifuge at 4℃12,000rpm (~13,400xg) for 30sec, and discard the waste liquid in the collection tube;

[0081] f. Add 500 μl of deproteinized solution RD to the adsorption column CR3 (please check whether ethanol has been added before use), centrifuge at 4°C 12,000 rpm (~13,400Xg) for 30 seconds, discard the waste liquid, and place CR3 in a collection tube;

[0082] g. Add 500 μl of rinse solution RW to the adsorption column CR3 (please check whether ethanol has been added first), let stand at room temperature for 2 min, centrifuge at 4°C 12,000 rpm (~13,400Xg) for 30 sec, and discard the waste liquid;

[0083] h. Repeat step 8;

[0084] i. Place the adsorption column in a 2 ml collection tube and centrifuge at 12,000 rpm (~13,400Xg) at 4°C for 2 min to remove the residual liquid;

[0085] j. Transfer the adsorption column CR3 into a new 1.5 ml centrifuge tube, add 30-100 μl of RNase-Free ddH2O, place at room temperature for 2 min, and centrifuge at 4°C, 12,000 rpm (~13,400Xg) for 2 min.

[0086] k. Use the reverse transcription kit TAKARA6210A to reverse transcribe the extracted sample RNA into DNA and store it for future use.

[0087] From the above, it can be seen that the quadruple fluorescent quantitative PCR species detection reagent for the four viruses (COV, AIV, NDV, WNV) carried by four wild birds provided by the present invention has the advantages of high efficiency, sensitivity, and safety. It can not only play a good supplementary role in the results of first-generation sequencing, but also can effectively distinguish a variety of viruses carried by wild animals, and provide strong support for customs border epidemic prevention inspections.

Claims

1. A quadruple fluorescent quantitative PCR primer probe set for rapid detection of four bird pathogens, characterized by: The quadruple fluorescent quantitative PCR primer probe includes specific primers and probes for coronavirus, Newcastle disease virus, avian influenza virus H9, and West Nile virus, wherein the upstream primer of coronavirus is shown in SEQ ID NO:1, the downstream primer is shown in SEQ ID NO:2, and the probe is shown in SEQ ID NO:3; the upstream primer of Newcastle disease virus is shown in SEQ ID NO:4, the downstream primer is shown in SEQ ID NO:5, and the probe is shown in SEQ ID NO:6; the upstream primer of avian influenza virus H9 is shown in SEQ ID NO:7, the downstream primer is shown in SEQ ID NO:8, and the probe is shown in SEQ ID NO:9; the upstream primer of West Nile virus is shown in SEQ ID NO:10, the downstream primer is shown in SEQ ID NO:11, and the probe is shown in SEQ ID NO:

12.

2. Use of the quadruple fluorescent quantitative PCR primer probe set for rapid detection of four bird pathogens as described in claim 1 in detecting avian coronavirus, Newcastle disease virus, avian influenza virus H9, and West Nile virus.

3. The use of the quadruple fluorescent quantitative PCR primer probe set for rapid detection of four bird pathogens according to claim 2 in detecting avian coronavirus, Newcastle disease virus, avian influenza virus H9, and West Nile virus, characterized in that: The specific steps include: (1) Extract RNA from throat swabs, anal swabs, stool or tissue samples using an RNA extraction kit; (2) reverse transcribing the RNA extracted in step (1) into cDNA; (3) Using the cDNA obtained in step (2) as a template, using the primers and probes shown in SEQ ID NO: 1 to SEQ ID NO: 12 to perform fluorescent quantitative PCR detection, and determining the results based on the Ct value.

4. The use of the quadruple fluorescent quantitative PCR primer probe set for rapid detection of four bird pathogens according to claim 3 in detecting avian coronavirus, Newcastle disease virus, avian influenza virus H9, and West Nile virus, characterized in that: The reaction system for fluorescence quantitative PCR detection in step (3) is 20 μL of 2×Pro TaqHS Probe Premix II, 4 μL of cDNA, 1 μL of upstream and downstream primers of avian influenza virus H9, 1.2 μL of upstream and downstream primers of Newcastle disease virus and West Nile virus, 0.5 μL of probe of avian influenza virus H9, 0.6 μL of probe of coronavirus, Newcastle disease virus and West Nile virus, and sterile water is added to make up to 40 μL.

5. The use of the quadruple fluorescent quantitative PCR primer probe set for rapid detection of four bird pathogens according to claim 3 in detecting avian coronavirus, Newcastle disease virus, avian influenza virus H9, and West Nile virus, characterized in that: The reaction procedure of the fluorescence quantitative PCR detection in step (3) is 95°C pre-denaturation for 30s; 95°C denaturation for 5s, 56°C annealing and extension for 30s, and 40 cycles.

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