Kit, primer and probe combination for simultaneously detecting avian influenza virus, Newcastle disease virus and infectious bronchitis virus

Abstract

The invention relates to the field of virus detection, in particular to a kit, primer and probe combination for simultaneously detecting avian influenza virus, Newcastle disease virus and infectious bronchitis virus. The kit disclosed by the invention comprises a nucleic acid amplification reagent, wherein the nucleic acid amplification reagent contains primers as shown in SEQ ID NO: 1 to SEQ ID NO: 6 and probes as shown in SEQ ID NO: 7 to SEQ ID NO: 9. The invention designs three pairs of primers and probes aiming at three pathogens of avian influenza virus, Newcastle disease virus and infectious bronchitis virus, and the primers and probes have good sensitivity and specificity and do not have non-specific reaction.

Description

Field of the Invention

[0001] The invention relates to the field of virus detection, and in particular to a kit and a primer and probe combination for simultaneously detecting avian influenza virus, Newcastle disease virus and infectious bronchitis virus. Background Art

[0002] Avian influenza, Newcastle disease and infectious bronchitis are currently important diseases that affect poultry farming. They can cause chicken deaths, decreased egg production in laying hens, and reduced feed conversion rates in broilers. They are highly contagious. All three viruses can cause respiratory tract lesions in poultry, and are acute and highly contagious, which poses a huge threat to the normal development of my country's poultry industry. At present, avian influenza, Newcastle disease and infectious bronchitis have been widely prevalent in my country, especially in areas with intensive chicken farming, and the prevalent virus genotypes are diverse and complex. It is urgent to develop a rapid and sensitive identification and detection method.

[0003] At present, there are separate RT-PCR detection standards for avian influenza, Newcastle disease and infectious bronchitis in China. However, there is no molecular method that can distinguish these three diseases and ensure consistent detection sensitivity for the three diseases based on similar clinical symptoms. When the three pairs of primers and probes react in the same premix, the coupled optical signals will affect each other, resulting in reduced sensitivity of the detection method. How to solve the mutual influence of optical signals and ensure that the identification detection method can detect the target with high sensitivity and specificity is the key and difficulty of multiple molecular biological detection methods.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The primary invention objective of the present invention is to provide a kit and a primer and probe combination for simultaneously detecting avian influenza virus, Newcastle disease virus and infectious bronchitis virus.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides a kit for simultaneously detecting avian influenza virus, Newcastle disease virus and infectious bronchitis virus. The kit comprises a nucleic acid amplification reagent, wherein the nucleic acid amplification reagent contains primers as shown in SEQ ID NO:1 to SEQ ID NO:6 and probes as shown in SEQ ID NO:7 to SEQ ID NO:9.

[0008] Optionally, the nucleic acid amplification reagent further contains buffer D; buffer D is an aqueous solution containing 0.2-0.3 mol / L (NH4)2SO4, 0.25-0.35 mol / L Tris-HCl, 0.025-0.035 mol / L MgCl2, and 0.3%-0.8% by volume of Tween-20;

[0009] Optionally, buffer D is an aqueous solution containing 0.25 mol / L (NH4)2SO4, 0.3 mol / L Tris-HCl, 0.03 mol / L MgCl2, and 0.5% by volume of Tween-20.

[0010] Optionally, the volume ratio of the buffer D in the nucleic acid amplification reagent is 15% to 25%, preferably 20%.

[0011] Optionally, the nucleic acid amplification reagent also contains Taq enzyme, dNTPs, and MMLV reverse transcriptase.

[0012] Optionally, in a 25 μL nucleic acid amplification system, the nucleic acid amplification reagent contains: 5 μL buffer D, 0.25 μL Taq enzyme, 4 μL 2.5 mM dNTP, 0.5 μL MMLV reverse transcriptase, 0.6 μL each of the primers and probes shown in SEQ ID NO: 1 to SEQ ID NO: 9, and 6.65 μL depc water.

[0013] Optionally, the concentrations of the primers and probes shown in SEQ ID NO: 1 to SEQ ID NO: 9 are both 10 to 30 pmol / L, preferably 20 pmol / L.

[0014] Optionally, in a 25 μL nucleic acid amplification system, the amount of template added is 5 μL.

[0015] Optionally, the kit also includes standard substances for preparing a standard curve, which are positive control fragments of avian influenza virus, Newcastle disease virus and infectious bronchitis virus, respectively. The nucleotide sequences of the positive control fragments are shown in SEQ ID NO: 10 to SEQ ID NO: 12.

[0016] Optionally, the kit further includes a negative control and a positive control, the negative control is deionized water, and the positive control is a positive control fragment having a nucleotide sequence as shown in SEQ ID NO: 10 to SEQ ID NO: 12.

[0017] The present invention provides a primer and probe combination for simultaneously detecting avian influenza virus, Newcastle disease virus and infectious bronchitis virus. The primer and probe combination comprises primers as shown in SEQ ID NO:1 to SEQ ID NO:6 and probes as shown in SEQ ID NO:7 to SEQ ID NO:9.

[0018] The technical solution provided by the embodiment of the present invention has the following advantages compared with the prior art:

[0019] Firstly, the present invention designs three pairs of primers and probes for three pathogens, namely, avian influenza virus, Newcastle disease virus and infectious bronchitis virus, which have good sensitivity and specificity and have no non-specific reaction with each other.

[0020] In the preferred technical solution of the present invention, a buffer D that can increase sensitivity is added to the premix system, thereby reducing the influence between detection primers and improving the sensitivity of the method. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The experimental results of the sensitivity of the avian influenza M gene in Example 3;

[0022] Figure 2 The experimental results of Newcastle disease M gene sensitivity in Example 3;

[0023] Figure 3 The results of the sensitivity experiment of the N gene of avian infectious bronchitis virus in Example 3 are as follows;

[0024] Figure 4 The specific experimental results in Example 4;

[0025] Figure 5 The results of different primer-probe combinations for detecting avian influenza samples in Example 5 are shown in FIG. 1 : wherein combination 1 is a red curve, combination 2 is a blue curve, and combination 3 is a green curve;

[0026] Figure 6 The results of the detection of Newcastle disease samples by different primer-probe combinations in Example 5, wherein combination 4 is a red curve, combination 5 is a blue curve, and combination 6 is a green curve;

[0027] Figure 7 The test results of different primer-probe combinations for chicken infectious bronchitis samples in Example 5, wherein combination 7 is a red curve, combination 8 is a blue curve, and combination 9 is a green curve;

[0028] Figure 8 The experimental results of different amplification systems in Example 6 are obtained, the amplification system with the addition of buffer D is the blue curve, and the amplification system without the addition of buffer D is the red curve;

[0029] Fig. 9 The results of amplifying avian influenza samples with different premixes in Example 7;

[0030] Fig.10 This is a graph showing the results of amplifying Newcastle disease samples using different premixes in Example 7;

[0031] Fig.11 This is a graph showing the results of amplifying chicken infectious bronchitis samples using different premixes in Example 7. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the specification.

[0033] The embodiment of the present invention provides a kit for simultaneously detecting avian influenza virus, Newcastle disease virus and infectious bronchitis virus, comprising a nucleic acid amplification reagent containing primers and probes as shown in SEQ ID NO:1 to SEQ ID NO:9.

[0034] As a specific implementation of the present invention, the primer and probe combination is shown in Table 1:

[0035] Table 1

[0036]

[0037] The nucleic acid amplification reagent of the embodiment of the present invention also contains buffer D; buffer D contains: (NH4)2SO40.2~0.3mol / L, Tris-HCl 0.25~0.35mol / L, MgCl2 0.025~0.035mol / L aqueous solution, and 0.3%~0.8% Tween-20 by volume; buffer D can increase sensitivity, reduce the influence between the three pairs of detection primers, and improve the sensitivity of the method. Optionally, buffer D is an aqueous solution containing (NH4)2SO4 0.25mol / L, Tris-HCl0.3mol / L, MgCl2 0.03mol / L, and 0.5% Tween-20 by volume.

[0038] As a specific implementation of the embodiment of the present invention, the preparation method of buffer D is shown in Table 2:

[0039] Table 2

[0040]

[0041] In an improvement of the embodiment of the present invention, the volume ratio of the buffer D in the nucleic acid amplification reagent is 15% to 25%, preferably 18% to 22%, and specifically 20%.

[0042] In an improvement of the embodiment of the present invention, Taq enzyme, dNTPs, and MMLV reverse transcriptase are further added to the nucleic acid amplification reagent.

[0043] An improvement of the embodiment of the present invention, in a 20 μL system, the nucleic acid amplification reagent contains 5 μL of buffer D, 0.25 μL of Taq enzyme, 4 μL of 2.5 mM dNTP, 0.5 μL of MMLV reverse transcriptase, 0.6 μL of each of the primers and probes shown in SEQ ID NO: 1 to SEQ ID NO: 9, and 6.65 μL of depc water. The concentration of the primers and probes is 10 to 30 pmol / L, preferably 20 pmol / L.

[0044] An improvement of the embodiment of the present invention, the kit also includes standard substances for preparing the standard curve, which are positive control fragments of avian influenza virus, Newcastle disease virus and infectious bronchitis virus, and the nucleotide sequences of the positive control fragments are shown in SEQ ID NO:10 to SEQ ID NO:12.

[0045] In an improvement of the embodiment of the present invention, the kit further includes a negative control substance and a positive control substance, wherein the negative control substance is deionized water, and the positive control substance is a positive control fragment with a sequence as shown in SEQ ID NO:10 to SEQ ID NO:12.

[0046] Specifically, the avian influenza positive control fragment is shown in SEQ ID NO:10, the Newcastle disease positive control fragment is shown in SEQ ID NO:11, and the chicken infectious bronchitis positive control fragment is shown in SEQ ID NO:12.

[0047] The specific sequence of the positive control fragment is shown in Table 3.

[0048] Table 3

[0049]

[0050] The embodiment of the present invention also provides a detection method for simultaneously detecting avian influenza virus, Newcastle disease virus and infectious bronchitis virus, comprising the following steps:

[0051] S1. Nucleic acid extraction: Use a laboratory-verified commercial nucleic acid extraction kit to extract nucleic acid from throat swabs, palate cleft swabs, trachea, lungs, kidneys, fallopian tubes and other tissue samples, as well as negative and positive controls.

[0052] S2. Prepare PCR reagents: Dispense the PCR reaction solution into PCR reaction tubes at a rate of 20 μL / tube.

[0053] S3, adding samples: adding 5 μL of each extracted nucleic acid sample to the PCR reaction tube containing the PCR reaction solution and mixing;

[0054] S4, PCR amplification detection;

[0055] S5. Set the baseline and threshold, and judge the results. After the experiment, use the instrument software for analysis. The threshold setting principle is that the threshold line just exceeds the highest point of the normal negative control curve (irregular noise line). Select 6 to 15 cycle areas for the FAM baseline. Determine whether the experimental results are valid, and then make a result judgment.

[0056] The embodiment of the present invention also provides a primer and probe combination for simultaneously detecting avian influenza virus, Newcastle disease virus and infectious bronchitis virus. The nucleotide sequences of the primer and probe combination are shown in SEQ ID NO: 1 to SEQ ID NO: 9, respectively.

[0057] Example 1

[0058] This embodiment provides a kit for simultaneously detecting avian influenza virus, Newcastle disease virus and infectious bronchitis virus. The composition of a single detection system in the kit is shown in Table 4:

[0059] Table 4

[0060]

[0061] The primer sequences are shown in Table 1 and were synthesized by Shanghai Bioengineering Co., Ltd. The composition of buffer D is shown in Table 2. Taq enzyme, MMLV Reverse Transcriptase and 2.5 mM dNTP mixture were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd., and (NH4)2SO4, MgCl2, and Tween-20 were purchased from Sigma.

[0062] Example 2

[0063] Usage of the kit of the present invention and method for determining the results:

[0064] 1. Nucleic acid extraction (performed in the sample processing area)

[0065] It is recommended to use a laboratory-verified commercial nucleic acid extraction kit to extract nucleic acid from pharyngeal swabs, cleft palate swabs, trachea, lungs, kidneys, fallopian tubes and other tissue samples, as well as negative and positive controls.

[0066] 2. PCR reagent preparation (performed in the reagent preparation area)

[0067] The PCR reaction solution was dispensed into PCR reaction tubes at a rate of 20 μL / tube, and the reaction tubes containing the PCR reaction solution were moved to the sample processing area.

[0068] 3. Sample addition (in the sample processing area)

[0069] Use a pipette tip with a filter to take 5 μL of each extracted nucleic acid sample and add them to the PCR reaction tubes containing the PCR reaction solution. Cover the tubes, vortex and centrifuge for a few seconds, and then move to the amplification detection area.

[0070] 4. PCR amplification detection (performed in the amplification detection area)

[0071] 4.1 Place the PCR reaction tube into the fluorescent PCR instrument and set the PCR instrument using the following parameters:

[0072] Reaction volume: 25 μL;

[0073] The detection channel selection is shown in Table 5:

[0074] Table 5

[0075] Target aisle AIV FAM ND VIC IBV CY5

[0076] The cycle program settings are shown in Table 6:

[0077] Table 6

[0078]

[0079]

[0080] 5. Baseline and threshold setting

[0081] After the experiment, the instrument software was used for analysis. The threshold setting principle was that the threshold line just exceeded the highest point of the normal negative control curve (irregular noise line). The FAM baseline selected 6 to 15 cycle areas.

[0082] 6. Conditions for the results to be established

[0083] The conditions for the results to be established are shown in Table 7:

[0084] Table 7

[0085] Reference FAM channel CY5 channel HEX / VIC Channel Negative control Not detected Not detected Not detected Positive Control Ct value ≤ 30 Ct value ≤ 30 Ct value ≤ 30

[0086] 7. Result determination

[0087] The results are shown in Table 8:

[0088] Table 8

[0089]

[0090] Embodiment 3:

[0091] This example is used to illustrate the sensitivity of the kit of the present invention:

[0092] The experimental materials are: 3 recombinant plasmids containing avian influenza M gene, Newcastle disease M gene, and chicken infectious bronchitis virus N gene were diluted to 5×10 5 , 5×10 4 , 5×10 3 , 5×10 2 , 5×10 1 copies / mL; the kit of Example 1 and the method of Example 2 were used for detection, and the experimental results obtained were as follows:

[0093] The experimental results of avian influenza M gene sensitivity are as follows Figure 1 And as shown in Table 9 (FAM channel):

[0094] Table 9

[0095]

[0096]

[0097] The experimental results of Newcastle disease M gene sensitivity are as follows Figure 2 And as shown in Table 10 (VIC channel):

[0098] Table 10

[0099]

[0100] Results of the sensitivity test of N gene of infectious bronchitis virus in chickens Figure 3 And as shown in Table 11 (CY5 channel):

[0101] Table 11

[0102]

[0103] According to the above experimental results, the analytical sensitivity of the kit and the detection method of the present invention is 5×10 2 copies / mL.

[0104] Embodiment 4:

[0105] This example is used to illustrate the specific experimental results:

[0106] The kit of Example 1 and the method of Example 2 were used to detect samples of avian bursal disease virus, avian reovirus, Mycoplasma gallisepticum, and Mycoplasma synoviae virus. The experimental results are as follows: Figure 4 shown.

[0107] according to Figure 4 It can be seen from the experimental results that the experimental results are all negative, and the specificity of the kit of the present invention is good.

[0108] Example 5

[0109] This example is used to illustrate the process and results of a primer or probe screening experiment:

[0110] 1. The kit of Example 1 and the method of Example 2 were used for detection, with the only difference being that the primer-probe combination 1 was replaced by combination 2 and combination 3, as shown in Table 12.

[0111] Table 12

[0112]

[0113] The above primer-probe combination was used to detect avian influenza samples from different sources, and the experimental results are shown in Table 13 and Figure 5 shown.

[0114] Table 13

[0115]

[0116] Comparing the curve shapes and Ct values ​​of the above experimental results, combination 1 has the highest sensitivity.

[0117] 2. The kit of Example 1 and the method of Example 2 were used for detection, with the only difference being that primer-probe combination 4 was replaced by combination 5 and combination 6, as shown in Table 14.

[0118] Table 14

[0119]

[0120] The above primer-probe combination was used to detect Newcastle disease samples from different sources, and the experimental results are shown in Table 15 and Figure 6 shown.

[0121] Table 15

[0122]

[0123] Comparing the curve shapes and Ct values ​​of the above experimental results, combination 4 has the highest sensitivity.

[0124] 3. The kit of Example 1 and the method of Example 2 were used for detection, with the only difference being that primer-probe combination 7 was replaced by combination 8 and combination 9, as shown in Table 16.

[0125] Table 16

[0126]

[0127] The above primer-probe combination was used to detect Newcastle disease samples from different sources, and the experimental results are shown in Table 17 and Figure 7 shown.

[0128] Table 17

[0129]

[0130] Comparing the curve shapes and Ct values ​​of the above experimental results, combination 7 has the highest sensitivity.

[0131] Example 6

[0132] This example is used to illustrate the effect of buffer D on amplification effect:

[0133] An amplification system without buffer D was prepared, and the specific composition is shown in Table 18:

[0134] Table 18

[0135]

[0136] Using the above amplification volume and the amplification system of Example 1, the positive control and SPF chicken tissue grinding liquid were tested (the method is the same as Example 2), and the experimental results obtained are shown in Tables 19 and Figure 8 shown.

[0137] Table 19

[0138]

[0139]

[0140] According to the above experimental results, the addition of buffer D reduced the CT value, improved the analytical sensitivity, and did not change the specificity.

[0141] Example 7

[0142] This example is used to compare the amplification effects of different premixes:

[0143] The commercial premix A used a multiplex probe one-step qPCR-RT kit (including UDC), Code: AQ322-01, purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0144] The commercial premix B used 5×Neoscript Fast RT Premix-UNG (probe qRT-PCR), Code: M5244-043801, which was purchased from Zhuhai Baorui Biotechnology Co., Ltd.

[0145] The reaction system of Example 1 of the present invention and the above two commercial premixes were used to test different samples (the method is the same as that of Example 2), and the experimental results obtained are shown in Table 20:

[0146] Table 20

[0147]

[0148] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those 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 described herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kit for simultaneously detecting avian influenza virus, Newcastle disease virus and infectious bronchitis virus, characterized in that: The kit includes a nucleic acid amplification reagent, which contains primers shown in SEQ ID NO:1 to SEQ ID NO:6 and probes shown in SEQ ID NO:7 to SEQ ID NO:

9.

2. The kit according to claim 1, characterized in that The nucleic acid amplification reagent also contains buffer D; The buffer D is an aqueous solution containing (NH4)2SO4 0.2-0.3mol / L, Tris-HCl 0.25-0.35mol / L, MgCl2 0.025-0.035mol / L, and 0.3%-0.8% by volume of Tween-20; Optionally, the buffer D is an aqueous solution containing 0.25 mol / L (NH4)2SO4, 0.3 mol / L Tris-HCl, 0.03 mol / L MgCl2, and 0.5% by volume of Tween-20.

3. The kit according to claim 2, characterized in that The volume ratio of the buffer D in the nucleic acid amplification reagent is 15% to 25%, preferably 20%.

4. The kit according to claim 1, characterized in that The nucleic acid amplification reagent also contains Taq enzyme, dNTPs and MMLV reverse transcriptase.

5. The kit according to claim 1, characterized in that In a 25 μL nucleic acid amplification system, the nucleic acid amplification reagent contains: 5 μL buffer D, 0.25 μL Taq enzyme, 4 μL 2.5 mM dNTP, 0.5 μL MMLV reverse transcriptase, 0.6 μL each of the primers and probes shown in SEQ ID NO: 1 to SEQ ID NO: 9, and 6.65 μL depc water.

6. The kit according to claim 5, characterized in that The concentrations of the primers and probes shown in SEQ ID NO: 1 to SEQ ID NO: 9 are all 10 to 30 pmol / L, preferably 20 pmol / L.

7. The kit according to claim 5, characterized in that In a 25 μL nucleic acid amplification system, the amount of template added is 5 μL.

8. The kit according to any one of claims 1 to 7, characterized in that The kit also includes standard substances for preparing a standard curve, which are positive control fragments of avian influenza virus, Newcastle disease virus and infectious bronchitis virus, and the nucleotide sequences of the positive control fragments are shown in SEQ ID NO: 10 to SEQ ID NO:

12.

9. The kit according to any one of claims 1 to 7, characterized in that The kit also includes a negative control and a positive control, wherein the negative control is deionized water, and the positive control is a positive control fragment with a nucleotide sequence as shown in SEQ ID NO: 10 to SEQ ID NO:

12.

10. A primer and probe combination for simultaneous detection of avian influenza virus, Newcastle disease virus and infectious bronchitis virus, characterized in that: The primer and probe combination includes primers as shown in SEQ ID NO:1 to SEQ ID NO:6 and probes as shown in SEQ ID NO:7 to SEQ ID NO:9.

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