CN110628954B

CN110628954B - H9N2 subtype avian influenza virus duplex fluorescence RT-LAMP detection primer group, kit and application

Info

Publication number: CN110628954B
Application number: CN201911054487.0A
Authority: CN
Inventors: 谢芝勋; 李云燕; 李孟; 黄娇玲; 罗思思; 李丹; 谢丽基; 曾婷婷; 张民秀; 张艳芳; 范晴; 谢志勤; 邓显文
Original assignee: Guangxi Veterinary Research Institute
Current assignee: Guangxi Veterinary Research Institute
Priority date: 2019-10-31
Filing date: 2019-10-31
Publication date: 2023-01-24
Anticipated expiration: 2039-10-31
Also published as: CN110628954A

Abstract

The invention discloses a detection primer group, a kit and application of H9N2 subtype avian influenza virus double fluorescence RT-LAMP, wherein the primer group comprises a primer 1-1, a primer 1-2, a primer 1-3, a primer 1-4, a primer 2-1, a primer 2-2, a primer 2-3 and a primer 2-4, and further comprises two probe primers 1-5 and a primer 2-5. According to the invention, the specific LAMP primers and the two specific probe primers are respectively designed for conserved sequences of HA and NA, so that the amplification reaction HAs very high specificity, the LAMP detection method is established after reaction conditions are optimized, the detection result is accurate and reliable, and meanwhile, the operation is simple, only one temperature-controllable water bath kettle is needed, so that the kit is suitable for rapid detection in basic aquatic veterinary stations and farms, and HAs a good application prospect.

Description

H9N2 subtype avian influenza virus duplex fluorescence RT-LAMP detection primer group, kit and application

Technical Field

The invention belongs to the technical field of molecular biology, and relates to a double fluorescence RT-LAMP detection primer group of H9N2 subtype avian influenza virus, a kit and application.

Background

Avian Influenza Virus (AIV) belongs to the family orthomyxoviridae, the genus of a type a virus. Avian influenza a viruses are classified into 16 HA subtypes and 9 NA subtypes according to their Hemagglutinin (HA) and Neuraminidase (NA) surface glycoproteins, but the tenth NA subtype and 17, 18 HA subtypes have been found in bats in the last two years. The H9 subtype AIV was first discovered in the united states since 1966 and has now caused very serious economic losses to the global poultry industry. In 1992, the first H9N2 epidemic situation is outbreak in Guangdong province in south China, which leads to large-area death and reduced laying rate of broiler chickens, and the host range of H9N2 is wide such as chickens, ducks, pigeons and the like. H9 subtype AIV also infects humans across species barriers, and since the first finding in guangdong of 1998 that humans are infected with H9N2 subtype AIV, the occurrence of H9N2 type AIV infection in humans is also ongoing. The H9 subtype avian influenza virus is widely existed at present, the common N2 subtype is combined to occur, the mutation potential is high, and the internal gene can be provided for other avian influenza subtypes which infect people, for example, H9N2 provides 6 internal gene segments for novel recombinant viruses H7N9 and H10N8, H5N1 influenza virus which infects people and is outbreaked in 1997 is also found to contain the internal gene of the H9N2 influenza virus subtype, therefore, H9N2 has great threat to the public health safety of poultry industry and human.

The current detection methods of the H9N2 virus mainly comprise: 1. PCR amplification is carried out by using traditional specific primers and then the PCR amplification is sent to a company for sequencing analysis, and the method is most reliable but long in time consumption; 2. the specific probe is designed, and the real-time RT-PCR method is utilized to analyze the result by monitoring the generation amount of fluorescence in real time through an instrument, so that the method has short detection time compared with the traditional PCR method, but needs a special instrument and is not suitable for detecting H9N2 virus by basic farmers. Therefore, there is an urgent need to establish a method for detecting the target gene, which is efficient, sensitive, rapid and simple

Loop-mediated isothermal amplification (LAMP) is an economical, rapid and sensitive method for amplifying viral nucleic acid under isothermal conditions, does not need special instruments, and has the advantages of low cost, visual result and the like. At present, no related report for establishing a method for detecting H9N2 by a single-double fluorescence reverse transcription isothermal amplification technology exists at home and abroad, and if a method for detecting H9N2 subtype avian influenza virus by using an LAMP technology can be developed, the method is very favorable for basic popularization.

Disclosure of Invention

The invention aims to provide a double fluorescence RT-LAMP detection primer group for H9N2 subtype avian influenza virus, a kit and application.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows: the invention provides a group of detection primer groups of double fluorescence RT-LAMP of H9N2 subtype avian influenza virus, which comprise 1-1 primer, 1-2 primer, 1-3 primer, 1-4 primer, 1-5 primer, 2-1 primer, 2-2 primer, 2-3 primer, 2-4 primer and 2-5 primer.

The sequence of the primer 1-1 is shown as SEQ ID NO.1, the sequence of the primer 1-2 is shown as SEQ ID NO.2, the sequence of the primer 1-3 is shown as SEQ ID NO.3, the sequence of the primer 1-4 is shown as SEQ ID NO.4, the sequence of the primer 1-5 is shown as SEQ ID NO.5, the sequence of the primer 2-1 is shown as SEQ ID NO.6, the sequence of the primer 2-2 is shown as SEQ ID NO.7, the sequence of the primer 2-3 is shown as SEQ ID NO.8, the sequence of the primer 2-4 is shown as SEQ ID NO.9, and the sequence of the primer 2-5 is shown as SEQ ID NO. 10;

wherein, the primers 1-5 and 2-5 are probe primers, the 5 'end of the primers 1-5 has FAM group, the 3' end has BHQ2 group, the 5 'end of the primers 2-5 has CY5 group, and the 3' end has BHQ2 group.

Reagents and kits containing the primer sets also belong to the protection scope of the invention.

The invention provides application of the primer group in detecting H9N2 subtype avian influenza virus.

The application further comprises the following steps:

(1) Extracting RNA of a sample to be detected, and then carrying out reverse transcription to obtain cDNA;

(2) Establishing an LAMP reaction system containing the primer group to carry out LAMP reaction;

(3) And (3) observing the detection result at 520nm and 670nm by using a multicolor fluorescence imaging system, and indicating that the H9N2 subtype avian influenza virus exists in the sample when yellow fluorescence is visible in the reaction tube.

Wherein, the LAMP reaction system is as follows: 3.5. Mu.L dNTP Mix (10 mmol/L, final concentration of 1.4mmol/L each), 2.5. Mu.L 10 XBst buffer, 1. Mu.L Bst DNA polymerase 8U (final concentration of 320U/L), 2. Mu.L MgSO ₄ (100 mmol/L, final concentration 8 mmol/L), 2. Mu.L of primer premix (final concentration of each primer in the system is primer 1-1 and primer 2-11.6. Mu. Mol/L, primer 1-2 and primer 2-2.6. Mu. Mol/L, primer 1-3 and primer 2-3, primer 1-4 and primer 2-4 0.2. Mu. Mol/L), 1. Mu.L of primer 1-5 and primer 2-5 probe premix (final concentration in the reaction system is primer 1-50.02. Mu. Mol/L, primer 2-5.02. Mu. Mol/L), 2. Mu.L of mixed cDNA template, and water is added to 25. Mu.L.

Wherein, the LAMP reaction condition is that the reaction is carried out for 80min at 60 ℃ and the inactivation is carried out for 5min at 80 ℃.

The invention provides application of the primer group and a reagent or kit containing the primer group in preparation of products for detecting and/or assisting in detecting whether a sample to be detected contains H9N2 subtype avian influenza virus.

The invention provides an application of a reagent or a kit containing the primer group in detecting H9N2 subtype avian influenza virus.

The invention has the advantages of

According to the invention, a specific LAMP primer and two specific probe primers are respectively designed for conserved sequences of HA and NA, so that the amplification reaction HAs high specificity, and the LAMP detection method is established after reaction conditions are optimized. The detection method can be completed only by reacting in a water bath kettle at 60 ℃ for 80 minutes, can specifically detect the H9N2 subtype avian influenza virus, HAs very high detection sensitivity, and can detect a 35 copy number of H9N2 HA and NA mixed plasmid sample. In conclusion, the primer and the method have the characteristics of being more specific and more sensitive than a conventional detection method, the visible double detection of the H9N2 subtype avian influenza virus is really realized through two fluorescent colors, the detection result is accurate and reliable, and meanwhile, the operation is simple, only one temperature-controllable water bath kettle is needed, so that the primer and the method are suitable for rapid detection in basic aquatic veterinary stations and farms, and have better application prospects.

Drawings

FIG. 1 shows the result of detecting the specificity of a primer set by RT-LAMP;

FIG. 2 is a sensitivity test of the detection system on the amplification curve of the LAMP real-time turbidimeter;

FIG. 3 shows the results of the sensitivity test of the detection system in a dual channel multicolor fluorescence imaging system;

FIG. 4 shows the results of a sensitivity test of a detection system in a 520nm channel of a multicolor fluorescence imaging system;

FIG. 5 shows the sensitivity of the detection system in the 670nm channel of a multicolor fluorescence imaging system;

FIG. 6 shows the results of an interference test of the detection system in a two-channel multicolor fluorescence imaging system;

FIG. 7 shows the results of interference testing of the detection system in a multi-color fluorescence imaging system with a 520nm channel;

FIG. 8 shows the interference test results of the detection system in the 670nm channel of the multicolor fluorescence imaging system.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting thereof. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and scope of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 primer design

Designing RT-LAMP detection primers according to HA and NA gene conserved sequences of H9N2 subtype avian influenza viruses in GenBank, wherein the RT-LAMP detection primers comprise 1-1 primer, 1-2 primer, 1-3 primer, 1-4 primer, 1-5 primer, 2-1 primer, 2-2 primer, 2-3 primer, 2-4 primer and 2-5 primer.

Example 2 detection of H9N2 subtype avian influenza Virus

1. The test samples and sources are shown in the following table:

TABLE 1 name and source of the specific test strains to be tested

A. Guangxi Zhuang autonomous region veterinary research institute B, university of Pennsylvania C, university of hong Kong D, university of Connecticut

2. Extracting RNA from a sample by using an RNA extraction kit, and then carrying out reverse transcription to obtain cDNA;

3. optimization of RT-LAMP reaction system, reaction conditions and construction of kit

Firstly, the following LAMP reaction system was established: 1-4. Mu.L dNTP Mix (10 mmol/L, final concentration of 0.4mmol/L-1.6 mmol/L), 2.5. Mu.L 10 XBst buffer, 1. Mu.L Bst DNA polymerase 8U (final concentration of 320U/L), 0.5-2.5. Mu.L MgSO ₄ (100 mmol/L, final concentration of 2mmol/L-10 mmol/L), 2. Mu.L of primer mixture (final concentration of each primer is 1-1 and 2-1.6. Mu. Mol/L, 1-2 and 2-2.6. Mu. Mol/L, 1-3 and 2-3, 1-4 and 2-4, 0.2. Mu. Mol/L), 1. Mu.L of primer 1-5 and 2-5 probe premix (final concentration is 1-5 0.02-0.05. Mu. Mol/L, 2-5.02-0.05. Mu. Mol/L), 2. Mu.L of mixed template (HA + NA plasmid), adding water to 25. Mu.L.

The reaction conditions are as follows: gradually reacting at 60 deg.C, 61 deg.C, 62 deg.C, 63 deg.C, 64 deg.C and 65 deg.C for 80min, and inactivating at 80 deg.C for 5min.

The following optimum reaction system and conditions were obtained by searching the above reaction system and conditions, respectively:

3.5. Mu.L dNTP Mix (10 mmol/L, final concentration of 1.4mmol/L each), 2.5. Mu.L 10 XBst buffer, 1. Mu.L Bst DNA polymerase 8U (final concentration of 320U/L), 2. Mu.L MgSO ₄ (100 mmol/L, final concentration 8 mmol/L), 2. Mu.L of primer premix (final concentration of each primer is 1-1 and 2-1.6. Mu. Mol/L, 1-2 and 2-21.6. Mu. Mol/L, 1-3 and 2-3, 1-4 and 2-4 0.2. Mu. Mol/L), 1. Mu.L of primer 1-5 and 2-5 probe premix (final concentration is 1-5.02. Mu. Mol/L, 2-5.02. Mu. Mol/L), 2. Mu.L of mixed cDNA template, and water is added to 25. Mu.L.

The LAMP reaction conditions are that the reaction is carried out at 60 ℃ for 80min and the reaction is carried out at 80 ℃ for 5min.

And (3) observing the detection result by using a multicolor fluorescence imaging system, wherein the detection result is shown in figure 1, and the detection result is observed in a 520nm and 670nm double channel, green fluorescence can be seen in the HA probe positive result reaction tube marked with FAM fluorescent group under the 520nm channel, and red fluorescence can be seen in the positive reaction tube by the NA probe marked with CY5 fluorescent group under the 670nm channel. Therefore, when yellow fluorescence is observed in the reaction tube, both channels are positive, and H9N2 subtype avian influenza virus exists in the sample.

In fig. 1, the tube samples are:

sample 1;

sample 2;

sample 3;

sample 4;

sample 5, RNA of A/Chicken/NY/273874/03 (H7N 2);

sample 6A/Duck/Guangxi/030D/2009 (H1N 1);

sample 7;

sample 8;

sample 9, A/Chicken/QT35/98 (H5N 9) RNA;

sample 10A/Duck/42846/07 (H7N 7) RNA;

sample 11A/Turkey/Ontario/6118/68 (H8N 4);

sample 12;

sample 13A/Duck/Guangxi/170/14 (H11);

sample 14;

sample 15;

sample 16;

sample 17A/Chiken/Guangxi/DX/2008 (H9N 2);

sample 18A/Quail/Guangxi/241B 5/2018 (H9N 2);

sample 19 (H9N 2);

sample 20A/Pheasent/Guangxi/222B 7/2018 (H9N 2);

sample 21A/Turtledove/Guangxi/191B 5/2017 (H9N 2);

sample 22A/Dove/Guangxi/211B 17-2/2017 (H9N 2);

sample 23A/Gull/Md/704/77 (H13N 5);

sample 24A/Mallard/Astrakhan/263/82 (H14N 5);

sample 25A/Shearwater/Australia/2576/79 (H15N 9);

sample 26;

sample 27;

sample 28;

sample 29, iltv;

sample 30 negative control

As can be seen from fig. 1, samples No.1, 2, 3, 4, and 5 exhibited red fluorescence; the sample No. 16 shows green, and the sample No.2 contains the avian influenza virus subtype H9;

samples

17, 18, 19, 20, 21 and 22 fluoresced yellow indicating the presence of H9N2 subtype avian influenza virus, and these results are consistent with the sample source.

4. Sensitivity detection

The H9N2-HA and H9N2-NA plasmids were diluted by 10 fold, 3.5X 10 ⁶ 3.5 copies/. Mu.L as a template, and LAMP reaction was carried out in accordance with the optimal reaction system and optimal reaction conditions in the above 3.

The results are shown in FIGS. 2-5, in which FIG. 2 is the amplification curve generated under LAMP real-time turbidimeter detection, FIGS. 3, 4 and 5 are the results of multicolor fluorescence imaging system, FIG. 3 is the results of two-channel observation, FIG. 4 is the results under 520nm channel, and FIG. 5 is the results under 670nm channel. Wherein 1: 3.5X 10 ⁶ Copy/. Mu.L; 2: 3.5X 10 ⁵ Copy/. Mu.L; 3: 3.5X 10 ⁴ Copy/. Mu.L; 4: 3.5X 10 ³ Copy/. Mu.L; 5: 3.5X 10 ² Copy/. Mu.L; 6:35 copies/. Mu.L; 7:3.5 copies/. Mu.L; 8: and (5) negative control. As can be seen, the minimum detection limit of LAMP on HA and NA gene plasmid results is 3.5 × 10 ¹ Copy/. Mu.L.

5. Interference detection

To verify whether high concentration of template would inhibit amplification of low concentration template, plasmid combinations of 8 different concentrations of HA and NA genes were prepared, as follows

(1)HA(7.5×10 ⁹ Copies/. Mu.L) + NA (7.5X 10 ⁵ Copy/. Mu.L);

(2)HA(7.5×10 ⁹ copies/. Mu.L) + NA (7.5X 10 ⁴ Copy/. Mu.L);

(3)HA(7.5×10 ⁹ copies/. Mu.L) + NA (7.5X 10 ³ Copy/. Mu.L);

(4)HA(7.5×10 ⁹ copies/. Mu.L) + NA (7.5X 10 ² Copy/. Mu.L);

(5)NA(7.5×10 ⁹ copies/. Mu.L) + HA (7.5X 10 ⁵ Copy/. Mu.L);

(6)NA(7.5×10 ⁹ copy/. Mu.L) + HA (7.5X 10 ⁴ Copy/. Mu.L);

(7)NA(7.5×10 ⁹ copies/. Mu.L) + HA (7.5X 10 ³ Copy/. Mu.L);

(8)NA(7.5×10 ⁹ copy/. Mu.L) + HA (7.5X 10 ² Copy/. Mu.L).

The results are shown in FIGS. 6 to 8; FIG. 6 shows the results of two-channel observation at 520nm and 670nm, wherein all 8 samples show yellow fluorescence; FIG. 7 shows the 520nm channel observation that all 8 samples showed green fluorescence; FIG. 8 shows the 670nm channel observation result, and all 8 samples show red fluorescence. As can be seen, the high concentration template and the low concentration template in different combinations do not interfere with each other during the reaction.

Sequence listing

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Claims

1. A detection primer group of H9N2 subtype avian influenza virus double fluorescence RT-LAMP is characterized by comprising 1-1 of primer, 1-2 of primer, 1-3 of primer, 1-4 of primer, 1-5 of primer, 2-1 of primer, 2-2 of primer, 2-3 of primer, 2-4 of primer and 2-5 of primer;

wherein, the primers 1-5 and 2-5 are probe primers, the 5 'end of the primers 1-5 is provided with FAM group, the 3' end is provided with BHQ2 group, the 5 'end of the primers 2-5 is provided with CY5 group, and the 3' end is provided with BHQ2 group.

2. A reagent or a kit comprising the primer set according to claim 1.

3. The primer group or the kit containing the primer group in claim 1 is applied to preparation of products for detecting or assisting in detecting whether a sample to be detected contains H9N2 subtype avian influenza virus.