阅读说明：本技术 一种引物组、探针、rpa试纸条试剂盒、鉴别方法 (Primer group, probe, RPA test strip kit and identification method ) 是由 沈永义 彭金玉 陈瑞爱 沈雪娟 于 2019-11-18 设计创作，主要内容包括：本发明属于病毒株鉴别方法技术领域,具体为一种用于检测非洲猪瘟病毒野毒株的引物组和探针,包括第一引物组和第一探针；所述第一引物组中的上游引物如序列SEQ ID NO：1所示；所述第一引物组中的下游引物为5’端带有生物素的如序列SEQ ID NO：2所示的DNA分子；所述第一探针为5’端带有羧基荧光素、3’端带有C3-spacer基团的如序列SEQ ID NO：3所示的DNA分子。同时,还提供了一种用于检测MGF360-505R基因缺失株的引物组和探针；该引物组和探针组特异性强、灵敏度高,同时,本发明还提出了基于RPA-LFD技术和上述引物组和探针组的RPA试纸条试剂盒和鉴别方法。(The invention belongs to the technical field of virus strain identification methods, and particularly relates to a primer group and a probe for detecting a wild strain of African swine fever virus, which comprise a first primer group and a first probe; the upstream primer in the first primer group is shown as a sequence SEQ ID NO: 1 is shown in the specification; the downstream primer in the first primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 2; the first probe is a probe which is provided with carboxyfluorescein at the 5 'end and C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 3. Meanwhile, a primer group and a probe for detecting MGF360-505R gene deletion strains are also provided; the primer group and the probe group have strong specificity and high sensitivity, and simultaneously, the invention also provides an RPA test strip kit and an identification method based on the RPA-LFD technology and the primer group and the probe group.)

1. A primer group and a probe for detecting a wild strain of African swine fever virus are characterized by comprising a first primer group and a first probe;

the upstream primer in the first primer group is shown as a sequence SEQ ID NO: 1 is shown in the specification;

the downstream primer in the first primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 2;

the first probe is a probe which is provided with carboxyfluorescein at the 5 'end and C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 3.

2. A primer group and a probe for detecting MGF360-505R gene deletion strains are characterized by comprising a second primer group and a second probe;

the upstream primer in the second primer group is shown as a sequence SEQ ID NO: 4 is shown in the specification;

the downstream primer in the second primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 5, a DNA molecule shown in the figure;

the second probe is a probe which is provided with carboxyfluorescein at the 5 'end and C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 6.

3. An RPA test strip kit for distinguishing African swine fever virus wild strains from MGF360-505R gene deletion strains is characterized by comprising: a first primer set and a first probe, a second primer set and a second probe;

the upstream primer in the first primer group is shown as a sequence SEQ ID NO: 1 is shown in the specification;

the downstream primer in the first primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 2;

the first probe is a probe which is provided with carboxyfluorescein at the 5 'end and C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 3, a DNA molecule shown in seq id no;

the upstream primer in the second primer group is shown as a sequence SEQ ID NO: 4 is shown in the specification;

the downstream primer in the second primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 5, a DNA molecule shown in the figure;

the second probe is a probe which is provided with carboxyfluorescein at the 5 'end and C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 6.

4. The RPA test strip kit for distinguishing the African swine fever virus wild strain from the MGF360-505R gene deletion strain as claimed in claim 3, further comprising a DNA extraction reagent, an RPA amplification reagent and a lateral flow test strip.

5. The RPA test strip kit for distinguishing the African swine fever virus wild strain from the MGF360-505R gene deletion strain as claimed in claim 3, further comprising magnesium acetate and ddH ₂O、5×Extration Buffer。

6. A rapid distinguishing method for identifying African swine fever virus wild strains and MGF360-505R gene deletion strains by an RPA-LFD technology is characterized by comprising the following steps:

s1: extracting viral DNA from the sample;

s2: placing the extracted virus DNA into an RPA reaction system by using the first primer group and the first probe, and the second primer group and the second probe respectively as described in claim 3 to perform RPA reaction, and obtaining amplification products A1 and A2 respectively

S3, fully reacting the amplification products A1 and A2 in the step S2 with the 5 × extraction Buffer respectively to obtain products B1 and B2;

s4, respectively dripping the products B1 and B2 obtained in the step 3 into a sample adding hole of the lateral flow test strip as claimed in claim 3, carrying out LFD reaction, observing the result, and determining the virus type.

7. The method according to claim 6, wherein step S2 is specifically:

taking 2.1 mu L of each of the upstream primer and the downstream primer in the first primer group, wherein the concentration of the upstream primer and the concentration of the downstream primer are 10 mu mol/L; 0.6 mu L of first probe with the concentration of 10 mu mol/L, 29.5 mu L of reduction Buffer, 12.2 mu L of ddH2O 12.2.2 mu L of RNase Inhibitor and 1 mu L of DNA template, adding the mixture into an RPA enzyme tube after mixing, shaking and uniformly mixing, adding 2.5 mu L of magnesium acetate, and carrying out initial reaction to obtain an amplification product A1;

taking 2.1 mu L of each of the upstream primer and the downstream primer in the second primer group, wherein the concentration of the upstream primer and the concentration of the downstream primer are 10 mu mol/L; 0.6 mu L of second probe with the concentration of 10 mu mol/L, 29.5 mu L of reduction Buffer, 12.2 mu L of ddH2O 12.2.2 mu L of RNase Inhibitor and 1 mu L of DNA template are mixed, added into an RPA enzyme tube and uniformly shaken, and then 2.5 mu L of magnesium acetate is added to start the reaction to obtain an amplification product A2.

8. The method according to claim 6, wherein in the step S3, the volume of the amplification product A1 and the amplification product A2 is 10 μ L; the 5 × extraction Buffer was 80 μ L.

9. The method of claim 6, wherein in step S4, the volume of products B1 and B2 is 75 μ L, and the result is read after the products are loaded into the loading wells for at least 5 min.

10. The method according to claim 6, wherein in step S4, the RPA enzyme tube comprises the recombinase uvs X, a single-strand binding protein, and a DNA polymerase; the RPA reaction conditions in step S2 are: the reaction system is placed in a water bath kettle at 37 ℃ for reaction for 20min without high-temperature denaturation, annealing and extension.

Technical Field

The invention relates to the technical field of virus strain identification methods, in particular to a primer group, a probe, an RPA test strip kit and an identification method.

Background

African Swine Fever (ASF) is an acute and highly-contagious infectious disease of domestic pigs and wild pigs caused by African Swine Fever Virus (ASFV), is clinically characterized by high fever, loss of appetite and bleeding of skin and internal organs, generally dies 2-10 days after illness, and the death rate can reach 100% in severe cases. The disease belongs to a group A animal epidemic disease which must be reported by the requirements of the world animal health Organization (OIE), and is classified as an animal epidemic disease in China. African swine fever is prevalent in tens of countries in africa, europe and america, causing significant economic losses and has spread to many asian countries bordering europe.

Since the first African swine fever outbreaks in our country in 2018, the African swine fever causes significant loss to the pig industry in China, and the healthy development of the pig industry in China is seriously influenced. Domestic and foreign researches show that after the African swine fever virus MGF360-505R gene is knocked out, the virus toxicity can be obviously reduced, and the African swine fever live virus with the two knocked-out genes is expected to become a vaccine. At present, CN201910348878.7 and CN 201910700685.3 respectively disclose a gene-deleted attenuated African swine fever virus which can be used as a vaccine, the vaccine and a construction method thereof, and the virulence gene of the African swine fever virus is deleted by a genetic engineering technology to obtain the gene-deleted virus with MGF360-505R deletion and combined deletion of CD2V and MGF 360-505R. Experiments show that the two strains can provide 100 percent immune protection for the Chinese epidemic virulent strain of the African swine fever, can be used as a safe and effective vaccine for preventing and controlling the epidemic situation of the Chinese African swine fever, and has great social value. However, the use of the vaccine can lead the result that the conventional method for detecting the African swine fever virus is jun positive, and the result that the African swine fever virus is positive due to wild virus infection or positive due to vaccination cannot be distinguished.

At present, ASF diagnosis methods include direct immunofluorescence experiments, indirect immunofluorescence experiments, methods for detecting viral nucleic acids and the like. Among them, the nucleic acid detection method is widely used because of its simple operation and high sensitivity. The Recombinase Polymerase Amplification (RPA) technique can accurately detect ASFV in a shorter time than these conventional detection techniques. RPA technology relies primarily on three enzymes: recombinase (recombination), single-stranded binding protein, and DNA polymerase. The whole RPA reaction process is very fast, and a large amount of target products can be amplified after the reaction is carried out for 20 minutes at a constant 37 ℃. The RPA reaction product can be rapidly detected by a flow-through immunodipstick (LFD). The RPA-LFD technology has the advantages of strong specificity, simple operation process, no initial heating step, high sensitivity, rapid detection and the like, and provides a possibility for detecting the African swine fever in the field.

Therefore, the technical problem to be solved by the application is as follows: how to provide a primer group, a probe, an RPA test strip kit and an identification method for identifying African swine fever virus wild strains and MGF360-505R gene deletion strains based on an RPA-LFD technology.

Disclosure of Invention

The invention aims to provide an RPA detection primer group and a probe group for distinguishing an African swine fever virus wild strain and an MGF360-505R gene deletion strain, the primer group and the probe group have strong specificity and high sensitivity, and meanwhile, the invention also provides an RPA test strip kit and an identification method based on the RPA-LFD technology and the primer group and the probe group.

Unless otherwise specified, all the% and parts in the present invention are weight percentages and parts, and M represents mol/L.

In order to achieve the purpose, the invention provides the following technical scheme:

a primer group and a probe for detecting a wild strain of African swine fever virus comprise a first primer group and a first probe;

the upstream primer in the first primer group is shown as a sequence SEQ ID NO: 1, the upstream primer we named: ASFV-MGF 360-505R-F1;

ASFV-MGF360-505R-F1：5’TGTGCTATTGCCCATAAGGATCTACATCTAT3’(SEQ ID NO：1)；

the downstream primer in the first primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 2; this downstream primer we named: ASFV-MGF 360-505R-R1;

ASFV-MGF360-505R-R1：5’Biotin-CATACTCAGAATGCCTATTATATTTGTTGAATTG 3’(SEQ ID NO：2)；

the first probe is a probe which is provided with carboxyl fluorescein at the 5 'end and C3-spacer group at the 3' end and has the sequence shown in SEQ ID NO: 3. The probe is named ASFV-MGF 360-505R-P1;

ASFV-MGF360-505R-P1：5’[FAM]TAACAGAATCGTACCCGATAAGTATCATCA[THF]TTTAGATATTCGCAT[C3-spacer]3’(SEQ ID NO：3)；

wherein biotin represents biotin, FAM represents carboxyfluorescein, THF represents a tetrahydrofuran linker, and C3-spacer means 3 methylene groups for preventing chain extension.

Meanwhile, the invention provides a primer group and a probe for detecting MGF360-505R gene deletion strains, which comprise a second primer group and a second probe;

the upstream primer in the second primer group is shown as a sequence SEQ ID NO: 4 is shown in the specification; the upstream primer is named as ASFV-MGF360-505 RQS-F2;

ASFV-MGF360-505RQS-F2：5’TATTTAATCATTTAGAGAAGGTCATCATAGGAG 3’(SEQ IDNO：4)；

the downstream primer in the second primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 5, a DNA molecule shown in the figure; the downstream primer is named as ASFV-MGF360-505 RQS-R2;

ASFV-MGF360-505RQS-R2：5’Biotin-CAGGATACGATTCACTACAATAGTGAGTAC 3’(SEQID NO：5)；

the second probe is a probe which has a carboxyl fluorescein at the 5 'end and a C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 6; this second probe we named ASFV-MGF360-505 RQS-P2;

ASFV-MGF360-505RQS-P2:5’[FAM]TTCAACGAGCAGGAAACAACTGTGTGCTTA[THF]TACAGCAACATACCC[C3-space r]3’(SEQ ID NO：6)。

wherein biotin represents biotin, FAM represents carboxyfluorescein, THF represents a tetrahydrofuran linker, and C3-spacer means 3 methylene groups for preventing chain extension.

Meanwhile, the invention provides an RPA test strip kit for distinguishing African swine fever virus wild strains from MGF360-505R gene deletion strains, which comprises:

a first primer set and a first probe, a second primer set and a second probe;

the upstream primer in the first primer group is shown as a sequence SEQ ID NO: 1 is shown in the specification;

the downstream primer in the first primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 2;

the first probe is a probe which is provided with carboxyl fluorescein at the 5 'end and C3-spacer group at the 3' end and has the sequence shown in SEQ ID NO: 3, a DNA molecule shown in seq id no;

the upstream primer in the second primer group is shown as a sequence SEQ ID NO: 4 is shown in the specification;

the downstream primer in the second primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 5, a DNA molecule shown in the figure;

the second probe is a probe which has a carboxyl fluorescein at the 5 'end and a C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 6.

In addition, the kit further comprises: a positive control and a negative control;

preferably, the positive control is plasmid DNA containing African swine fever virus MGF360-505R gene; the negative control is deionized water.

The RPA test strip kit for distinguishing the African swine fever virus wild strain from the MGF360-505R gene deletion strain further comprises a DNA extraction reagent, an RPA amplification reagent and a lateral flow test strip.

The RPA test strip kit for distinguishing the African swine fever virus wild strain from the MGF360-505R gene deletion strain further comprises magnesium acetate and ddH ₂O、5×Extration Buffer。

Finally, the invention also provides a rapid distinguishing method for identifying the African swine fever virus wild strain and the MGF360-505R gene deletion strain by the RPA-LFD technology, which comprises the following steps:

s1: extracting viral DNA from the sample;

s2: placing the extracted virus DNA into an RPA reaction system by using the first primer group and the first probe, and the second primer group and the second probe respectively as described in claim 3 to perform RPA reaction, and obtaining amplification products A1 and A2 respectively

S3, fully reacting the amplification products A1 and A2 in the step S2 with the 5 × extraction Buffer respectively to obtain products B1 and B2;

s4, respectively dripping the products B1 and B2 obtained in the step 3 into the sample adding holes (P1 and P2 test strips) of the lateral flow test strip of claim 3, carrying out LFD reaction, observing the result and determining the virus type.

And respectively dripping the amplification products B1 and B2 onto the sample adding holes of the test strip, wherein the detection results are as follows:

1) when the test strip detection area of the P1 shows color, the quality control area also shows color; the detection area of P2 is colored, and the quality control area is also colored, so that the virus in the sample to be detected is the mixed infection of wild strains and MGF360-505R gene deletion strains;

2) when the test strip detection area of the P1 shows color, the quality control area also shows color; the detection area of P2 does not develop color, and the quality control area develops color, so that the virus in the sample to be detected is infected by wild strains;

3) when the test strip detection area of the P1 strip does not develop color, and the quality control area develops color; the detection area of P2 is developed, and the quality control area is also developed, so that the virus in the sample to be detected is MGF360-505R gene deletion strain;

4) when the test strip detection area of the P1 strip does not develop color, and the quality control area develops color; and (3) the detection area of the P2 does not develop color, and the quality control area develops color, so that the sample to be detected does not contain viruses.

5) When the quality control area does not develop color, the test strip is ineffective, and a new test strip is needed for re-determination.

In the above method, step S2 specifically includes:

taking 2.1 mu L of each of the upstream primer and the downstream primer in the first primer group, wherein the concentration of the upstream primer and the concentration of the downstream primer are 10 mu mol/L; 0.6 mu L of first probe with the concentration of 10 mu mol/L, 29.5 mu L of reduction Buffer, 12.2 mu L of ddH2O 12.2.2 mu L of RNase Inhibitor and 1 mu L of DNA template, adding the mixture into an RPA enzyme tube after mixing, shaking and uniformly mixing, adding 2.5 mu L of magnesium acetate, and carrying out initial reaction to obtain an amplification product A1;

taking 2.1 mu L of each of the upstream primer and the downstream primer in the second primer group, wherein the concentration of the upstream primer and the concentration of the downstream primer are 10 mu mol/L; 0.6 mu L of second probe with the concentration of 10 mu mol/L, 29.5 mu L of reduction Buffer, 12.2 mu L of ddH2O 12.2.2 mu L of RNase Inhibitor and 1 mu L of DNA template are mixed, added into an RPA enzyme tube and uniformly shaken, and then 2.5 mu L of magnesium acetate is added to start the reaction to obtain an amplification product A2.

In the above method, in the step S3, the volumes of the amplification product a1 and the amplification product a2 are 10 μ L; the 5 × extraction Buffer was 80 μ L.

In the above method, in the step S4, the volume of the products B1 and B2 is 75 μ L, and the result is read after the sample is loaded into the sample loading well for at least 5 min.

In the above method, in step S4, the RPA enzyme tube contains recombinase uvs X, single-strand binding protein, and DNA polymerase; the RPA reaction conditions in step S2 are: the reaction system is placed in a water bath kettle at 37 ℃ for reaction for 20min without high-temperature denaturation, annealing and extension. .

Compared with the prior art, the invention has the beneficial effects that:

1. recombinase Polymerase Amplification (RPA) is a method of amplifying a target fragment using a specific primer pair and a probe under isothermal conditions. The entire process is carried out very quickly and detectable levels of amplification product are typically obtained within ten minutes. The flow measurement immune test strip technology (LFD) is used for carrying out test strip detection on an RPA amplification product, the detection result of the amplification product on a lateral flow test strip can be observed within a few minutes, complex instruments and equipment are not needed, and the LFD is suitable for on-site rapid detection.

2. According to the project, the regions of the primers and the probes are skillfully designed, so that whether a detected sample is infected by the African swine fever virus or not and whether an infected strain has gene deletion or not can be simply, conveniently and rapidly identified, whether mixed infection of a wild strain and a gene deletion strain exists or not can be judged, and the result can be observed by naked eyes through detection of a test strip.

3. The method has the advantages of simple operation, strong specificity, high sensitivity and the like, only generates specific amplification reaction on the DNA of African Swine Fever Virus (ASFV), and has no amplification reaction on nucleic acids of swine fever virus (CSFV), porcine pseudorabies virus (PRV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Parvovirus (PPV) and the like.

4. The kit provides a new means for detecting the African swine fever virus in China, is convenient and fast to apply and operate clinically, has strong practicability, can be used for epidemic situation monitoring, differential diagnosis and epidemic disease purification of the African swine fever virus in production practice, can also be used for rapid identification of the African swine fever virus strain in a professional laboratory, and can provide technical support for improving the comprehensive prevention and control level of the African swine fever virus in China.

Drawings

FIG. 1 shows the results of African Swine Fever Virus (ASFV) RPA-LFD assay of example 2.

FIG. 2 shows the results of electrophoretic validation of the African Swine Fever Virus (ASFV) RPA product of example 2.

FIG. 3 shows the results of the African Swine Fever Virus (ASFV) RPA-LFD specificity test of example 3.

FIG. 4 shows the results of electrophoresis of the African Swine Fever Virus (ASFV) RPA product specificity of example 3.

FIG. 5 shows the results of African Swine Fever Virus (ASFV) RPA-LFD susceptibility assay of example 4.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.