CN113337643A

CN113337643A - RT-LAMP primer group and kit for detecting bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus

Info

Publication number: CN113337643A
Application number: CN202110722948.8A
Authority: CN
Inventors: 李占鸿; 杨恒; 廖德芳; 杨振兴; 李卓然; 李华春
Original assignee: Yunnan Animal Science and Veterinary Institute
Current assignee: Yunnan Animal Science and Veterinary Institute
Priority date: 2021-06-28
Filing date: 2021-06-28
Publication date: 2021-09-03

Abstract

The invention relates to an RT-LAMP primer group and a kit for detecting bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus, which comprise a primer group I, a primer group II and a primer group III; the primer group I consists of BTV-F3, BTV-B3, BTV-FIP, BTV-BIP, BTV-LB and BTV-LF; the primer group II consists of EHDV-F3, EHDV-B3, EHDV-FIP, EHDV-BIP, EHDV-LB and EHDV-LF; the primer group III consists of PALV-F3, PALV-B3, PALV-FIP, PALV-BIP and PALV-LF. The RT-LAMP detection method has high specificity and sensitivity, can quickly and accurately detect BTV, EHDV and PALV, and has great popularization value.

Description

RT-LAMP primer group and kit for detecting bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus

Technical Field

The invention belongs to the technical field of veterinary infectious disease detection, and particularly relates to an RT-LAMP primer group and a kit for visually detecting bluetongue virus, animal epidemic hemorrhagic disease virus and palinuria virus.

Background

Bluetongue virus (BTV), Epidemic Hemorrhagic Disease Virus (EHDV), and peliema virus (Palyam virus, PALV) are ruminant arboviruses transmitted by culicoides and belong to members of the circovirus genus of the reoviridae family. The genomes of BTV, EHDV and PALV are composed of 10 segments (Seg-1-Seg-10) of Double-stranded RNA (dsRNA), the size is about 20kb, and the genes have closer genetic relationship in genetic evolution.

BTV infected sheep can cause significant clinical symptoms, manifested as hyperpyrexia, wasting, congestion, swelling and erosion of lips, bleeding and ulceration of nasal and gastrointestinal mucosa, lameness due to crown abscission, etc.; when new serum type BTV is introduced, the incidence rate of infected sheep can reach 80%, the mortality rate can reach 40%, the normal international trade of cattle and sheep livestock products is seriously influenced, and huge economic loss is caused to the cattle and sheep breeding industry, so the BT is classified as a legal report animal epidemic disease by the world animal health Organization (OIE), and the BT is classified as a first-class animal epidemic disease in China. EHDV has stronger pathogenicity to deer and cattle, and clinical symptoms are mainly shown as follows: the body temperature is increased, shock, bleeding and erosion of oral cavity and abomasum mucous membrane occur, hoof crown bleeding and lameness occur, abortion or dead fetus occurs to the pregnant animals, and the milk yield of the dairy cows or the lactating animals is reduced sharply, so that the EHD is listed as a cross-border animal epidemic disease reported by OIE in the statutory. Infection of ruminants such as cattle and sheep with PALV can cause abnormal production of livestock in gestation period, manifested by abortion, dead fetus or abnormal fetus with hypoplasia of brain tissue.

The method has the advantages of wide range of members in China, complex and various natural climate conditions and wide range of activity of vector insects, and provides favorable conditions for the propagation of the bovine and sheep arboviruses throughout the country. At present, 27 BTV serotypes (BTV-1 to BTV-27) are confirmed worldwide, and the prevalence of 12 BTV serotypes (BTV-1, -2, -3, -4, -5, -7, -9, -12, -15, -16, -21 and-24) exists in China and is mainly distributed in provinces and cities such as Yunnan, Guangdong, Guangxi, Jiangsu, Hunan and Xinjiang. Currently, EHDV of 9 serotypes (EHDV-1, -2, -4, -5, -6, -7, -8, -9 and-10 types) is found all over the world, and the prevalence of 5 serotype strains such as EHDV-1, -5, -6, -7 and-10 exists in China, and is mainly distributed in provinces such as Yunnan, Guangdong, Guangxi and Nemeng. The prevalence of PALV in our country is spread over a wide range from south (hainan) to north (inner Mongolia) and from east (Jiangsu) to west (Xinjiang), and the circulating strains include three sera of zhongshan virus (Chuzan virus, CHUV), Bunyip Creek Virus (BCV) and D' Aguilar virus (DAV). The wide prevalence of BTV, EHDV and PALV in China poses serious threat to the health development of cattle and sheep breeding industry in China.

The susceptible hosts of BTV, EHDV and PALV are consistent, the clinical symptoms are similar, and the viruses have a closer genetic relationship, so that a rapid and accurate identification method is very necessary to be developed. At present, various nucleic acid detection methods such as RT-PCR and qRT-PCR are established for BTV, EHDV and PALV, but the methods need to use complex and expensive instruments such as a PCR instrument, so the methods are mainly limited to laboratory detection and cannot realize rapid field detection. Therefore, how to overcome the defects of the prior art is a problem which needs to be solved urgently in the technical field of veterinary infectious disease detection.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an RT-LAMP primer group and a kit for visually detecting bluetongue virus, animal epidemic hemorrhagic disease virus and palidia virus.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the primer group for detecting the bluetongue virus, the animal epidemic hemorrhagic disease virus and the Parieyamu virus comprises a primer group I, a primer group II and a primer group III;

the primer group I consists of a primer BTV-F3, a primer BTV-B3, a primer BTV-FIP, a primer BTV-BIP, a primer BTV-LB and a primer BTV-LF;

the primer BTV-F3 is (a1) or (a 2):

(a1) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 1;

(a2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the SEQ ID NO.1 and has the same function as the SEQ ID NO. 1;

the primer BTV-B3 is (a3) or (a 4):

(a3) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 2;

(a4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the SEQ ID NO.2 and has the same function as the SEQ ID NO. 2;

the primer BTV-FIP is (a5) or (a6) as follows:

(a5) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 3;

(a6) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID NO.3 and has the same function as SEQ ID NO. 3;

the primer BTV-BIP is (a7) or (a8) as follows:

(a7) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 4;

(a8) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the SEQ ID NO.4 and has the same function as the SEQ ID NO. 4;

the primer BTV-LF is (a9) or (a10) as follows:

(a9) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 5;

(a10) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the SEQ ID NO.5 and has the same function as the SEQ ID NO. 5;

the primer BTV-LB is (a11) or (a 12):

(a11) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 6;

(a12) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO.6 and has the same function as SEQ ID NO. 6;

the primer group II consists of a primer EHDV-F3, a primer EHDV-B3, a primer EHDV-FIP, a primer EHDV-BIP, a primer EHDV-LB and a primer EHDV-LF;

the primer EHDV-F3 is (b1) or (b2) as follows:

(b1) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 7;

(b2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the SEQ ID NO.7 and has the same function as the SEQ ID NO. 7;

the primer EHDV-B3 is (B3) or (B4) as follows:

(b3) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 8;

(b4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the SEQ ID NO.8 and has the same function as the SEQ ID NO. 8;

the primer EHDV-FIP is (b5) or (b6) as follows:

(b5) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 9;

(b6) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the SEQ ID NO.9 and has the same function as the SEQ ID NO. 9;

the primer EHDV-BIP is (b7) or (b8) as follows:

(b7) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 10;

(b8) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the SEQ ID NO.10 and has the same function as the SEQ ID NO. 10;

the primer EHDV-LF is (b9) or (b10) as follows:

(b9) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 11;

(b10) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO.11 and has the same function as SEQ ID NO. 11;

the primer EHDV-LB is (b11) or (b12) as follows:

(b11) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 12;

(b12) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO.12 and has the same function as SEQ ID NO. 12;

the primer group III consists of a primer PALV-F3, a primer PALV-B3, a primer PALV-FIP, a primer PALV-BIP and a primer PALV-LF;

the primer PALV-F3 is (c1) or (c2) as follows:

(c1) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 13;

(c2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO.13 and has the same function as SEQ ID NO. 13;

the primer PALV-B3 is (c3) or (c4) as follows:

(c3) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 14;

(c4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO.14 and has the same function as SEQ ID NO. 14;

the primer PALV-FIP is (c5) or (c6) as follows:

(c5) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 15;

(c6) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO.15 and has the same function as SEQ ID NO. 15;

the primer PALV-BIP is (c7) or (c8) as follows:

(c7) a single-stranded DNA molecule shown in a sequence table SEQ ID NO. 16;

(c8) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO.16 and has the same function as SEQ ID NO. 16;

the primer PALV-LF is (c9) or (c10) as follows:

(c9) a single-stranded DNA molecule shown in SEQ ID NO. 17;

(c10) DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO.17 and has the same function with SEQ ID NO. 17;

in the primer group I, the molar ratio of BTV-F3, BTV-B3, BTV-FIP, BTV-BIP, BTV-LB and BTV-LF is specifically 1: 1: 3: 3: 2: 2. in the primer group II, the molar ratio of EHDV-F3, EHDV-B3, EHDV-FIP, EHDV-BIP, EHDV-LB and EHDV-LF can be 1: 1: 4: 4: 3: 3. in the primer group III, the molar ratio of the PALV-F3, the PALV-B3, the PALV-FIP, the PALV-BIP and the PALV-LF is specifically 1: 1: 4: 4: 3.

the primer combination is used in any one of the following (d1) to (d 6):

(d1) identifying bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus;

(d2) preparing a kit for identifying bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus;

(d3) identifying whether the viruses to be detected are bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus;

(d4) preparing a kit for identifying whether the virus to be detected is bluetongue virus or animal epidemic hemorrhagic disease virus or paliyama virus;

(d5) identifying whether the sample to be detected contains bluetongue virus and/or animal epidemic hemorrhagic disease virus and/or palivium virus;

(d6) preparing a kit for identifying whether the sample to be detected contains bluetongue virus and/or animal epidemic hemorrhagic disease virus and/or palivium virus.

The invention also protects a kit containing the primer combination; the application of the kit is (e1), (e2) or (e 3):

(e1) identifying bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus;

(e2) identifying whether the viruses to be detected are bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus;

(e3) and identifying whether the sample to be detected contains bluetongue virus and/or animal epidemic hemorrhagic disease virus and/or palivium virus.

The invention also provides a method for identifying whether the viruses to be detected are bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus, which comprises the following steps:

(1) extracting RNA of the virus to be detected, heating at 95 ℃ for 3min, immediately carrying out ice bath, and carrying out denaturation treatment;

(2) taking the viral RNA extracted in the step (1) as a template, respectively adopting a primer group I, a primer group II and a primer group III of the primer combination to carry out RT-LAMP amplification reaction, and judging according to the reaction result as follows:

if the primer group I can realize positive amplification by using the virus RNA as a template, the virus to be detected is BTV; if the positive amplification with the virus RNA as a template can be realized by adopting the primer group II, the virus to be detected is EHDV; if the positive amplification with the virus RNA as a template can be realized by adopting the primer group III, the virus to be detected is PALV.

In the method, the virus to be detected is BTV, EHDV or PALV.

The invention also provides a method for identifying whether a sample to be detected contains BTV and/or EHDV and/or PALV, which comprises the following steps:

(1) extracting total RNA of a sample to be detected, heating at 95 ℃ for 3min, immediately carrying out ice bath, and carrying out denaturation treatment;

(2) taking the total RNA of the sample to be detected extracted in the step (1) as a template, respectively adopting a primer group I, a primer group II and a primer group III of the primer combination to carry out RT-LAMP amplification reaction, and judging according to the reaction result as follows:

if the primer group I is adopted, the positive amplification with the total RNA of the sample to be detected as a template can be realized, and the sample to be detected contains BTV; if the primer group II is adopted, the positive amplification with the total RNA of the sample to be detected as a template can be realized, and the sample to be detected contains EHDV; if the primer group III is adopted, the positive amplification with the total RNA of the sample to be detected as a template can be realized, and the sample to be detected contains PALV; if the primer group I, the primer group II and the primer group III are not used for realizing positive amplification by taking the total RNA of the sample to be detected as a template, the sample to be detected does not contain BTV, EHDV and PALV.

In any of the above methods, the "positive amplification" can be specifically determined by the following method: after the reaction, the reaction solution is judged according to the color change, and positive amplification is performed if the color of the reaction solution changes from red to yellow, and negative amplification is performed if the color of the reaction solution remains the same as red.

The invention also protects the primer group I.

The application of the primer group I is (f1) or (f 2):

(f1) identifying whether the virus to be detected is BTV;

(f2) and identifying whether the sample to be tested contains BTV.

The invention also protects the application of the primer group I in the preparation of a kit A, and the kit A is used as the following (f1) or (f 2):

(g1) identifying whether the virus to be detected is BTV;

(g2) and identifying whether the sample to be tested contains BTV.

The invention also protects a kit A containing the primer group I, and the application of the kit A is as follows (f1) or (f 2):

(f1) identifying whether the virus to be detected is BTV;

(f2) and identifying whether the sample to be tested contains BTV.

The invention also protects the primer group II.

The application of the primer group II is (f3) or (f 4):

(f3) identifying whether the virus to be detected is EHDV;

(f4) and identifying whether the sample to be tested contains EHDV.

The invention also protects the application of the primer group II in the preparation of a kit B, and the application of the kit B is as follows (f3) or (f 4):

(f3) identifying whether the virus to be detected is EHDV;

(f4) and identifying whether the sample to be tested contains EHDV.

The invention also protects a kit B containing the primer group II, and the application of the kit B is as follows (f3) or (f 4):

(f3) identifying whether the virus to be detected is EHDV;

(f4) and identifying whether the sample to be tested contains EHDV.

The invention also protects the primer group III.

The application of the primer group III is (f5) or (f 6):

(f5) identifying whether the virus to be detected is PALV;

(f6) and identifying whether the sample to be tested contains the PALV.

The invention also protects the application of the primer group III in the preparation of a kit C, and the application of the kit C is as follows (f5) or (f 6):

(f5) identifying whether the virus to be detected is PALV;

(f6) and identifying whether the sample to be tested contains the PALV.

The invention also protects a kit C containing the primer group III, and the application of the kit C is as follows (f5) or (f 6):

(f5) identifying whether the virus to be detected is PALV;

(f6) and identifying whether the sample to be tested contains the PALV.

Any sample to be detected can be virus and cattle and sheep anticoagulated blood which are subjected to separation culture.

In any one of the above reaction systems for LAMP amplification by using the primer set I, the final concentration of the primer mixture in the primer set I is as follows: 0.2. mu.M BTV-F3, 0.2. mu.M BTV-B3, 0.6. mu.M BTV-FIP, 0.6. mu.M BTV-BIP, 0.4. mu.M BTV-LF, 0.4. mu.M BTV-LB.

In any one of the above reaction systems for LAMP amplification by using the primer group II, the final concentration of the primer mixture in the primer group II is as follows: 0.2. mu.M EHDV-F3, 0.2. mu.M EHDV-B3, 0.8. mu.M EHDV-FIP, 0.8. mu.M EHDV-BIP, 0.6. mu.M EHDV-LF, 0.6. mu.M EHDV-LB.

In any one of the above reaction systems for LAMP amplification by using the primer group III, the final concentration of the primer mixture in the primer group III is as follows: 0.2 μ M PALV-F3, 0.2 μ M PALV-B3, 0.8 μ M PALV-FIP, 0.8 μ M PALV-BIP, 0.6 μ M PALV-LF.

In any of the above methods, the loop-mediated isothermal amplification reaction conditions are: keeping the temperature at 64 ℃ for 45 min.

Any one of the reaction systems for RT-LAMP amplification by using the primer group I can specifically be as follows: WarmStart LAMP 2 × Master Mix 12.5 μ L, BTV-F31 μ L, BTV-B31 μ L, BTV-FIP 1 μ L, BTV-BIP 1 μ L, BTV-LF 1 μ L, BTV-LB 1 μ L, nucleic acid template 2 μ L, RNase-free water was added to make up to 25 μ L.

Any one of the reaction systems for RT-LAMP amplification by using the primer group II can specifically be as follows: WarmStart LAMP 2 × Master Mix 12.5 μ L, EHDV-F31 μ L, EHDV-B31 μ L, EHDV-FIP 1 μ L, EHDV-BIP1 μ L, EHDV-LF 1 μ L, EHDV-LB 1 μ L, nucleic acid template 2 μ L, RNase-free water was added to make up to 25 μ L.

The reaction system for performing RT-LAMP amplification by adopting the primer group III specifically comprises: WarmStart LAMP 2 × Master Mix 12.5 μ L, PALV-F31 μ L, PALV-B31 μ L, PALV-FIP 1 μ L, PALV-BIP 1 μ L, PALV-LF 1 μ L, nucleic acid template 2 μ L, RNase-free water was added to make up to 25 μ L.

The reaction procedure of any one of the RT-LAMP amplifications can be specifically as follows: keeping the temperature at 64 ℃ for 45 min. And (3) judging the result according to the color change of the reaction liquid after the reaction is finished: if the reaction solution changes from red to yellow, the amplification is positive, and if the reaction solution remains red, the amplification is negative.

Loop-mediated isothermal amplification (LAMP) adopted by the invention is an isothermal nucleic acid amplification technology, and Bst DNA polymerase with strand displacement activity and waterfall type nucleic acid amplification function is utilized to perform the denaturation and automatic circulation strand displacement nucleic acid amplification reaction of nucleic acid under the isothermal condition. Compared with other nucleic acid amplification detection technologies, the technology does not need expensive instruments and equipment, the reaction process can be completed in a constant-temperature water bath kettle, the operation is simple and convenient, and the technology has the advantages of high sensitivity, high specificity, high detection speed, low cost, visual result and the like, and is particularly suitable for detecting pathogens at the basement level or on site.

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

the invention provides an RT-LAMP primer and a method for detecting 3 cattle and sheep arbovirus. The 3 viruses include: BTV, EHDV, and PALV. The RT-LAMP detection method has high specificity and sensitivity; the reaction process can be carried out in a constant-temperature water bath kettle without complex instruments; the reaction time is short, and the reaction can be completed only in 45 min; the detection result can be judged by naked eyes, and the field visual detection of BTV, EHDV and PALV is realized; has great popularization value.

Drawings

FIG. 1 is a diagram showing experimental results established by BTV, EHDV and PALV RT-LAMP detection methods;

wherein: A. c, E is a visual result chart, B, D, F is an electrophoresis chart of RT-LAMP amplification products; A. b is a result chart established by the BTV RT-LAMP detection method, wherein the number 1 is BTV-9 type virus, the number 2 is a blood sample infected with BTV-9 type virus, and the number 3 is negative control; C. d is a result chart established by the EHDV RT-LAMP detection method, the number 1 is EHDV-10 type virus, the number 2 is a blood sample infected with the EHDV-10 type virus, and the number 3 is negative control; E. f is a result chart established by the PALV RT-LAMP detection method, the number 1 is CHUV virus, the number 2 is a blood sample infected with CHUV virus, and the number 3 is negative control; m is DNA Marker DL5000 (the size of a Marker strip is 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp in sequence).

FIG. 2 shows the results of BTV RT-LAMP sensitivity experiments;

wherein: a is a visual result diagram of RT-LAMP, and B is an electrophoresis diagram of RT-LAMP amplification products; the numbers 1-6 are 10-fold dilutions of BTV-Seg-5ssRNA to 4.5X 10⁵Copies/. mu.L to 4.5X 10⁰6 copied/mu L gradient samples, 7 as negative control, M as DNA Marker DL5000 (the size of Marker band is 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp in sequence).

FIG. 3 shows the results of EHDV RT-LAMP sensitivity experiments;

wherein: a is a visual result diagram of RT-LAMP, and B is an electrophoresis diagram of RT-LAMP amplification products; the numbers 1-6 are 10-fold dilutions of EHDV-Seg-5ssRNA to 6.2X 10⁵Copies/. mu.L to 6.2X 10⁰6 copied/mu L gradient samples, 7 as negative control, M as DNA Marker DL5000 (the size of Marker band is 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp in sequence).

FIG. 4 shows the result of the PALV RT-LAMP sensitivity experiment;

wherein: a is a visual result diagram of RT-LAMP, and B is an electrophoresis diagram of RT-LAMP amplification products; the number 1-6 is 10 times diluted by PALV-Seg-7ssRNA to 2.3X 10⁵Copy/. mu.L to 2.3X 10⁰6 copied/mu L gradient samples, 7 as negative control, M as DNA Marker DL5000 (the size of Marker band is 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp in sequence).

FIG. 5 is a diagram showing the results of BTV RT-LAMP;

wherein: a is a visual result diagram of RT-LAMP, and B is an electrophoresis diagram of RT-LAMP amplification products; the numbers 1-12 are sequentially amplification results of nucleic acid templates of 12 serotype BTV strains such as BTV-1, -2, -3, -4, -5, -7, -9, -12, -15, -16, -21 and-24, the numbers 13-17 are sequentially amplification results of EHDV-1, CHUV, AKAV, AHSV and FMDV total RNA serving as templates, 18 is negative control, and M is DNA Marker DL5000 (the size of a Marker band is 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp sequentially).

FIG. 6 is a graph showing the results of a specificity experiment of EHDV RT-LAMP;

wherein: a is a visual result diagram of RT-LAMP, and B is an electrophoresis diagram of RT-LAMP amplification products; the numbers 1-7 are sequentially amplification results with 7 serotype EHDV strain nucleic acids such as EHDV-1, -2, -5, -6, -7, -8 and-10 as templates, the numbers 8-12 are sequentially amplification results with BTV-1, CHUV, AKAV, AHSV and FMDV total RNA as templates, the number 13 is negative control, and the M is DNA Marker DL5000 (the size of a Marker strip is 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp sequentially).

FIG. 7 is a graph showing the results of the specificity experiment of PALV RT-LAMP;

wherein: a is a visual result diagram of RT-LAMP, and B is an electrophoresis diagram of RT-LAMP amplification products; the numbers 1-3 are amplification results with CHUV, BCV and DAV strain nucleic acids as templates, the numbers 4-8 are amplification results with BTV-1, EHDV-1, AKAV, AHSV and FMDV total RNA as templates, the number 9 is negative control, and the number M is DNA Marker DL5000 (the size of the Marker band is 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100 bp).

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.

Example 1 primer design and preparation

The BTV Seg-5 sequence, the EHDV Seg-5 sequence and the PALV Seg-7 sequence are downloaded from GenBanK, and are compared and analyzed by MEGA5.0, highly conserved regions are selected, and 3 sets of RT-LAMP amplification primers are designed by using Primer Exporer V5 online software.

The primer group for identifying BTV comprises two outer primers (BTV-F3 and BTV-B3), two inner primers (BTV-FIP and BTV-BIP) and two loop primers (BTV-LB and BTV-LF); the primer group for identifying the EHDV comprises two outer primers (EHDV-F3 and EHDV-B3), two inner primers (EHDV-FIP and EHDV-BIP) and two loop primers (EHDV-LB and EHDV-LF); the primer group for identifying the PALV comprises two outer primers (PALV-F3 and PALV-B3), two inner primers (PALV-FIP and PALV-BIP) and a loop primer (PALV-LF), and the sequences of the primers in each group are shown in Table 1.

TABLE 1 BTV, EHDV and PALV group-specific RT-LAMP detection primer information

The primers were synthesized by Shanghai Czeri bioengineering, Inc., and dissolved with RNase-free water.

Example 2 detection method establishment

1. Sample to be tested

Virus to be tested 1: BTV-9 strain and monitoring animal blood samples for virus isolation (described in Lizhanhong, Wangjinping, Yangheng, etc.. bluetongue virus serotype 9 strain was first isolated in our country [ J ]. animal veterinary bulletin, 2019,50(2): 354-363.).

Blood sample to be tested 1: sentinel cattle EDTA anticoagulated blood samples from which BTV-9 strains were isolated (described in Lizhanhong, Wangjinnu, Yangheng, et al. bluetongue virus serotype 9 strain was first isolated in our country [ J ]. veterinary bulletin of zootechnics, 2019,50(2): 354-.

Virus to be tested 2: the EHDV-10 strain (described in the following documents: Lizhanhong, Share, Yangxing, etc. bovine derived Epidemic Hemorrhagic Disease Virus (EHDV) serum 10 type strain is isolated and identified in China [ J ] Virus bulletin, 2019,35(01): 118-.

Blood sample to be tested 2: sentinel cattle EDTA anticoagulated blood samples from which EHDV-10 strains were isolated (described in Lizhanhong, Xiaolei, Yang Shaoxing, etc.. bovine derived Epidemic Hemorrhagic Disease Virus (EHDV) serotype 10 strains were isolated and identified in China [ J ]. Virol, 2019,35(01): 118-.

Virus to be tested 3: CHUV strains (described in Yangtze, Lihuachun, Sharey, et al, isolation and sequence characterization of Palimam serogroup viruses in southern China in 2012-2016 [ J ] Prov. veterinary Proc. 2018,49(4):761 770.).

Blood sample to be tested 3: sentinel bovine EDTA anticoagulated blood samples from which CHUV strains were isolated (described in Yanghen, Lihuachun, Share, et al, 2012 2016. isolation and sequence characterization of Palima serogroup Virus in southern China, [ J ]. Prov. veterinary Commission 2018,49(4): 761-770.).

Sample to be tested 4: RNase-free water.

2. Primary reagents and instruments

Used in this and the following examples

LAMP color-changing premix was purchased from New England Biolab Inc. (Beijing); the magnetic bead method virus nucleic acid extraction kit is purchased from Applied biosystems of America; nucleic acid automatic extractor KingFisher Flex from Applied biosystems, USA; constant temperature water bath is purchased from Shanghai Kesheng appearanceAppliance equipment, inc.

3. RT-LAMP detection was performed as follows

(1) Extracting total RNA of a sample to be detected, heating at 95 ℃ for 3min, immediately carrying out ice bath, and carrying out denaturation treatment.

(2) The total RNA of the virus 1 to be detected and the blood sample 1 to be detected denatured in the step (1) were taken as templates, RNase-free water was taken as a negative control, and RT-LAMP amplification was performed using the primer set I prepared in example 1.

Reaction system of LAMP amplification: WarmStart LAMP 2 × Master Mix 12.5 μ L, BTV-F31 μ L, BTV-B31 μ L, BTV-FIP 1 μ L, BTV-BIP 1 μ L, BTV-LF 1 μ L, BTV-LB 1 μ L, nucleic acid template 2 μ L, RNase-free water was added to make up to 25 μ L. The final concentration of each primer in the reaction system is as follows: 0.2. mu.M BTV-F3, 0.2. mu.M BTV-B3, 0.6. mu.M BTV-FIP, 0.6. mu.M BTV-BIP, 0.4. mu.M BTV-LF, 0.4. mu.M BTV-LB.

The reaction program of RT-LAMP amplification is specifically as follows: keeping the temperature at 64 ℃ for 45 min.

(3) The total RNA of the virus 2 to be detected and the blood sample 2 to be detected extracted in the step (1) are taken as templates, RNase-free water is taken as negative control, and the primer group II prepared in the embodiment 1 is adopted for RT-LAMP amplification.

Reaction system of RT-LAMP amplification: WarmStart LAMP 2 × Master Mix 12.5 μ L, EHDV-F31 μ L, EHDV-B31 μ L, EHDV-FIP 1 μ L, EHDV-BIP1 μ L, EHDV-LF 1 μ L, EHDV-LB 1 μ L, nucleic acid template 2 μ L, RNase-free water was added to make up to 25 μ L. The final concentration of each primer in the reaction system is as follows: 0.2. mu.M EHDV-F3, 0.2. mu.M EHDV-B3, 0.8. mu.M EHDV-FIP, 0.8. mu.M EHDV-BIP, 0.6. mu.M EHDV-LF, 0.6. mu.M EHDV-LB.

(4) The total RNA of the virus 3 to be detected and the blood sample 3 to be detected extracted in the step (1) are taken as templates, RNase-free water is taken as negative control, and the primer group III prepared in the embodiment 1 is adopted for RT-LAMP amplification.

Reaction system of RT-LAMP amplification: WarmStart LAMP 2 × Master Mix 12.5 μ L, PALV-F31 μ L, PALV-B31 μ L, PALV-FIP 1 μ L, PALV-BIP 1 μ L, PALV-LF 1 μ L, nucleic acid template 2 μ L, RNase-free water was added to make up to 25 μ L. The final concentration of each primer in the reaction system is as follows: 0.2 μ M PALV-F3, 0.2 μ M PALV-B3, 0.8 μ M PALV-FIP, 0.8 μ M PALV-BIP, 0.6 μ M PALV-LF.

(5) And (4) judging a result: and judging the result according to the color change of the reaction solution and the electrophoresis of the amplification product.

Taking the total RNA of the virus 1 to be detected and the blood sample 1 to be detected as templates, adopting a primer group I to carry out RT-LAMP amplification, changing the color of reaction liquid of the virus 1 to be detected and the blood sample 1 to be detected from red to yellow after the reaction is finished (figure 1A), and enabling the electrophoresis of an amplification product to present a gradient-shaped strip (figure 1B); while the reaction solution color of the negative control remained unchanged in red (FIG. 1A) and no amplified band was generated (FIG. 1B). The virus 1 to be detected is BTV, and the blood sample 1 to be detected contains BTV.

Taking the total RNA of the virus 2 to be detected and the blood sample 2 to be detected as templates, adopting a primer group II to carry out RT-LAMP amplification, changing the color of reaction liquid of the virus 2 to be detected and the blood sample 2 to be detected from red to yellow after the reaction is finished (figure 1C), and enabling the electrophoresis of an amplification product to present a gradient-shaped strip (figure 1D); while the reaction solution color of the negative control remained unchanged in red (FIG. 1C) and no amplified band was generated (FIG. 1D).

Performing RT-LAMP amplification by using total RNA of a virus 3 to be detected and a blood sample 3 to be detected as templates and adopting a primer group III, wherein after the reaction is finished, the color of reaction liquid of the virus 3 to be detected and the blood sample 3 to be detected is changed from red to yellow (figure 1E), and the electrophoresis of an amplification product presents a gradient-shaped strip (figure 1F); while the reaction solution color of the negative control remained red (FIG. 1E) and no amplification band was generated (FIG. 1F). The virus 3 to be detected is CHUV, and the blood sample 3 to be detected contains CHUV.

Example 3 sensitivity

1. Preparation of standards

The ssRNA template of the target gene is obtained by transcription with a T7 in vitro transcription kit, the concentration of the in vitro transcribed ssRNA is determined by using a NanoDrop 2000 nucleic acid concentration tester, and the nucleic acid copy number is calculated. The ssRNA with determined copy number was diluted in a 10-fold gradient as a standard.

2. Sensitivity testing

(1) The samples to be tested are respectively as follows: BTV-Seg-5ssRNA (copy number: 4.5X 10)¹⁰Copies/. mu.L), EHDV-Seg-5ssRNA (copy number: 6.2X 10¹⁰Copies/. mu.L) and CHUV-Seg-7ssRNA (copy number: 2.3X 10¹¹Copy/. mu.L);

(2) diluting a sample to be detected: using RNase-free water to perform 10-fold gradient dilution on each sample to be detected, and diluting BTV-Seg-5ssRNA to 4.5 multiplied by 10⁰Copies/. mu.L to 4.5X 10⁵Copy/. mu.L, EHDV-Seg-5ssRNA dilution to 6.2X 10⁰Copies/. mu.L to 6.2X 10⁵Copy/. mu.L, dilution of PALV-Seg-7ssRNA to 2.3X 10⁰Copy/. mu.L to 2.3X 10⁵Copies/. mu.L.

(3) Taking each diluted standard ssRNA obtained in the step (2) as a template, and detecting by the detection method of the embodiment 2.

The results showed that the copy number of BTV-Seg-5ssRNA was 4.5X 10⁰When the sample is copied/mu L, the color of the reaction solution changes from red to yellow (FIG. 2A), and the electrophoresis of the amplification product presents a gradient-shaped band (FIG. 2B); the reaction solution of the negative control remained red (FIG. 2A) and no amplification band was generated (FIG. 2B), indicating that the lower limit of BTV RT-LAMP detection for BTV-Seg-5ssRNA was 4.5 copies/. mu.L. EHDV-Seg-5ssRNA copy number 6.2X 10¹When the sample is copied/microliter, the color of the reaction solution changes from red to yellow (FIG. 3A), and the electrophoresis of the amplification product presents a gradient-shaped band (FIG. 3B); the reaction solution color of the negative control remained red (FIG. 3A) and no amplification band was generated (FIG. 3B), indicating that the lower limit of detection of EHDV RT-LAMP detection method for EHDV-Seg-5ssrNA was 62 copies/. mu.L. The PALV-Seg-7ssRNA is 2.3 multiplied by 10¹When the sample is copied/microliter, the color of the reaction solution changes from red to yellow (FIG. 4A), and the electrophoresis of the amplification product presents a gradient-shaped band (FIG. 4B); the color of the reaction solution of the negative control remained red (FIG. 4A) and no amplification band was generated (FIG. 4B), indicating that the detection limit of the PALV RT-LAMP detection method for PALV-Seg-7ssRNA was 23 copies/. mu.L.

Example 4 specificity

1. Sample to be examined

(1) 12 serotype BTV strains such as BTV-1, -2, -3, -4, -5, -7, -9, -12, -15, -16, -21 and-24 (described in the following documents: Lizhahong, Songzong, Yangxing, and the like, the establishment and application of a one-step RT-PCR typing method for twelve serotype bluetongue viruses popular in China [ J ] Chinese veterinary science, 2020,50(12): 1486-one 1493 ].

(2) 7 serotype EHDV strains (described in the following documents: Yangxing, Zhujiangbo, Lizhanhong, and the like, the establishment and application of a double fluorescent quantitative RT-PCR detection method for bluetongue virus and epidemic hemorrhagic disease virus [ J ] Chinese veterinary science, 2019,49(09):1104 + 1111 ]).

(3) 3 serotype strains of PALV such as CHUV, BCV and DAV (described in Yanheng, Lihuachun, Share, et al, 2012-2016. isolation and sequence characterization of the serotype of Palimam in southern China [ J ]. Proc. veterinary Commission on zootechnics, 2018,49(4): 761-770.).

(4) AKAV strains (described in the following documents: Lizhanhong, Yangxing, Lizhuang, etc.. the isolation and identification of the African plaque virus in the sentinel goats in Yunnan province and the retrospective analysis of its infection [ J ] Chinese veterinary medical bulletin 2021(02): 121-.

(5) FMDV inactivated vaccine strains were purchased from Qianhao organism, Inc.

(6) African Horse Sickness Virus (AHSV) inactivated vaccines are introduced from the world animal health Organization (OIE) reference laboratory (Onderstepopoort Veterinary Institute: South Africa).

The detection was carried out by the detection method of example 2.

2. Primary reagents and instruments

The main reagents and apparatus were identical to those of example 2.

3. RT-LAMP detection was performed as follows

(2) The BTV-1, -2, -3, -4, -5, -7, -9, -12, -15, -16, -21 and-2412 serotype BTV strains extracted in the step (1), EHDV-1, CHUV, AKAV, AHSV and FMDV total RNA are taken as templates, RNase-free water is taken as a negative control, and the primer group I prepared in the example 1 is adopted for RT-LAMP amplification.

(3) And (2) taking the EHDV-1, -2, -5, -6, -7, -8 and-107 serotype EHDV strains, BTV-1, CHUV, AKAV, AHSV and FMDV total RNA extracted in the step (1) as a template, taking RNase-free water as negative control, and carrying out RT-LAMP amplification by adopting the primer group II prepared in the example 1.

(4) Taking the total RNA of CHUV, BCV, DAV, BTV-1, EHDV-1, AKAV, AHSV and FMDV extracted in the step (1) as a template, taking RNase-free water as negative control, and adopting the primer group III prepared in the example 1 to carry out RT-LAMP amplification.

Adopting a primer group I to respectively carry out RT-LAMP detection on total RNA of 12 serotype BTV, EHDV-1, CHUV, AKAV, AHSV and FMDV strains such as BTV-1, -2, -3, -4, -5, -7, -9, -12, -15, -16, -21 and-24, and the like, after the reaction is finished, the color of a reaction solution corresponding to the RNA of the 12 serotype BTV strains such as BTV-1, -2, -3, -4, -5, -7, -9, -12, -15, -16, -21 and-24 is changed from red to yellow (figure 5A), and the electrophoresis of an amplification product presents a gradient-shaped band (figure 5B); the colors of the reaction solution of EHDV-1, CHUV, AKAV, AHSV and FMDV strains and negative control are kept unchanged in red (FIG. 5A), and no amplification band is generated (FIG. 5B), which indicates that the primer group I can specifically detect BTV, has no cross with other common bovine and ovine arboviruses, and has good specificity.

Performing RT-LAMP detection on the total RNA of 7 serotype EHDV strains such as EHDV-1, -2, -5, -6, -7, -8, 10 and the like, namely EHDV, BTV-1, CHUV, AKAV, AHSV and FMDV respectively by using a primer group II, wherein after the reaction is finished, the color of reaction liquid corresponding to the RNA of the 7 serotype EHDV strains such as EHDV-1, -2, -5, -6, -7, -8, 10 and the like is changed from red to yellow (figure 6A), and the electrophoresis of an amplification product presents a gradient strip (figure 6B); the reaction solution color of BTV-1, CHUV, AKAV, AHSV, FMDV strains and negative control is kept unchanged in red (FIG. 6A), and no amplification band is generated (FIG. 6B), which indicates that the primer group II can specifically detect EHDV, has no cross with other common bovine and ovine arboviruses, and has good specificity.

Performing RT-LAMP detection on total RNA of CHUV, BCV, DAV, BTV-1, EHDV-1, AKAV, AHSV and FMDV strains by using a primer group III, wherein after the reaction is finished, the color of reaction liquid corresponding to the CHUV, BCV and DAV strains RNA is changed from red to yellow (figure 7A), and the electrophoresis of an amplification product presents a gradient-shaped strip (figure 7B); the reaction solution color of BTV-1, EHDV-1, AKAV, AHSV and FMDV strains and negative control is kept unchanged in red (FIG. 7A), and no amplification band is generated (FIG. 7B), which indicates that the primer group III can specifically detect PALV, has no cross with other common cattle and sheep arboviruses, and has good specificity.

Example 5 application

1. Culture virus detection

(1) Virus to be tested 1: 46 strains of BTV of 12 serotypes isolated from provinces such as Yunnan, Guangxi, Guangdong and Jiangsu in 1996 to 2020 (Table 2);

TABLE 2 isolation information for 12 serotypes BTV isolated in China representing strains

^aSerotype	Separated from each other	Time of separation	^bNumber representing strain
				BTV-1(n＝5)	Yunnan and Guangxi provinces	1996、2012、2016、2020	V147
BTV-2(n＝4)	Yunnan and Guangdong	1996、1997、2013、2014	V146
				BTV-3(n＝5)	Yunnan province	1997、2012、2013、2014	V025
BTV-4(n＝5)	Yunnan, Guangxi and Guangdong	1996、2012、2016、2017	V030
				BTV-5(n＝4)	Yunnan and Guangdong	2012、2013、2015、2016	V084
BTV-7(n＝2)	Guangdong (Chinese character of Guangdong)	2014、2020	V089
				BTV-9(n＝2)	Yunnan province	2012、2015	V008
BTV-12(n＝4)	Yunnan, Guangxi and Guangdong	1997、2012、2014	V007
				BTV-15(n＝3)	Yunnan and Jiangsu provinces	1996、1997、2014	V061
BTV-16(n＝5)	Yunnan, Guangxi and Guangdong	1996、2012、2014、2017	V109
				BTV-21(n＝4)	Yunnan, Guangxi and Guangxi	1996、2013、2014、2015	V057
BTV-24(n＝3)	Yunnan province	2012、2013、2015	V015

^aNumbers in parentheses indicate the number of strains represented by each BTV serotype used in this study;

^beach serotype for RT-LAMP specificity validation represents a strain.

Virus to be tested 2: 31 EHDV strains were classified into 5 serotypes (EHDV-1, -5, -6, -7, and-10) (Table 3);

TABLE 3 isolation information for 5 serotypes EHDV representatives of the strains isolated in China

^aSerotype	Separated from each other	Time of separation	^bNumber representing strain
				EHDV-1(n＝6)	Yunnan, Guangxi and Guangdong	2013、2014、2016	V132
EHDV-5(n＝9)	Yunnan and Guangxi provinces	2013、2014、2015、2016	V023
				EHDV-6(n＝11)	Yunnan and Guangdong	2013、2014、2015、2016	V003
EHDV-7(n＝2)	Yunnan province	2013、2015	V269
				EHDV-10(n＝3)	Yunnan province	2013、2016、2017	V277

^aThe numbers in parentheses indicate the number of strains represented by each EHDV serotype used in this study;

^beach serotype for RT-LAMP specificity validation represents a strain.

Virus to be tested 3: the 21 strains of PALV belong to 3 serotypes, such as CHUV, BCV and DAV (Table 4).

TABLE 4 isolation information for 3 serotypes of PALV isolated in China representing strains

^aSerotype	Separated from each other	Time of separation	^bNumber representing strain
				CHUV(n＝14)	Yunnan province	2012、2014、2015、2020	V144
BCV(n＝4)	Yunnan province	2014、2015、2016	V104
				DAV(n＝3)	Yunnan province	2014、2015、2016	V106

^aThe numbers in parentheses indicate the number of strains represented by each of the PALV serotypes used in this study;

^beach serotype for RT-LAMP specificity validation represents a strain.

(2) Primary reagents and instruments

The main reagents and apparatus were the same as in example 2.

(3) RT-LAMP detection was performed as follows

1) Extracting total RNA of a sample to be detected, heating at 95 ℃ for 3min, immediately carrying out ice bath, and carrying out denaturation treatment.

2) RT-LAMP amplification was performed using the primer set I prepared in example 1, using RNA of 46 BTV strains extracted in step 1) as a template and RNase-free water as a negative control.

3) The RNA of 31 EHDV strains extracted in the step 1) is taken as a template, RNase-free water is taken as a negative control, and RT-LAMP amplification is carried out by adopting the primer group II prepared in the example 1.

The reaction program of RT-LAMP amplification is specifically as follows: keeping the temperature at 64 ℃ for 45 min. .

4) RT-LAMP amplification was performed using the primer set III prepared in example 1, using RNA of 21 strains of PALV extracted in step 1) as a template and RNase-free water as a negative control.

5) Determination of results

The primer group I is adopted to carry out RT-LAMP detection on RNA of 46 BTV strains respectively, after the reaction is finished, the color of reaction liquid of the 46 BTV strains is changed from red to yellow, and the color of reaction liquid of negative control is kept unchanged, so that the primer group I can effectively detect 12 serotype BTV Chinese epidemic strains separated in different time and different regions.

And (3) respectively carrying out RT-LAMP detection on the RNA of 31 EHDV strains by using a primer group II, wherein after the reaction is finished, the color of reaction liquid of the 31 strains is changed from red to yellow, and the color of reaction liquid of negative control is kept unchanged, so that the primer group II can effectively detect 5 serotype EHDV Chinese epidemic strains separated at different time and different regions.

And (3) respectively carrying out RT-LAMP detection on the RNA of the 21 strains of PALV by adopting a primer group III, wherein after the reaction is finished, the color of the reaction solution of the 21 strains is changed from red to yellow, and the color of the reaction solution of the negative control is kept unchanged, so that the primer group III can effectively detect the PALV Chinese epidemic strains separated at different time and different regions.

2. Positive blood sample detection

(1) Sample to be tested 1: 10 parts of 12 serotype BTV infected sentinel cattle and sheep BTV anticoagulated blood samples collected in 2013 to 2020, wherein the total amount is 120 parts;

clinical specimen to be examined 2: the total amount of the EDTA anticoagulation blood samples of the sentinel cattle infected by the 5 serotype EHDV collected from 2013 to 2017 is 40;

clinical specimen to be examined 3: the total of 21 PALV infected sentinel bovine EDTA anticoagulated blood samples collected from 2012 to 2020.

(2) Primary reagents and instruments

The main reagents and apparatus were the same as in example 2.

(3) Sample detection

2) mu.L of the total RNA of BTV-positive blood extracted in step 1) was used as a template, RNase-free water was used as a negative control, and RT-LAMP amplification was performed using the primer set I prepared in example 1.

Reaction system of RT-LAMP amplification: WarmStart LAMP 2 × Master Mix 12.5 μ L, BTV-F31 μ L, BTV-B31 μ L, BTV-FIP 1 μ L, BTV-BIP 1 μ L, BTV-LF 1 μ L, BTV-LB 1 μ L, nucleic acid template 4 μ L, RNase-free water was added to make up to 25 μ L. The final concentration of each primer in the reaction system is as follows: 0.2. mu.M BTV-F3, 0.2. mu.M BTV-B3, 0.6. mu.M BTV-FIP, 0.6. mu.M BTV-BIP, 0.4. mu.M BTV-LF, 0.4. mu.M BTV-LB.

3) BTV qRT-PCR detection was performed simultaneously with reference to literature reported methods (described in: establishment and application of a double fluorescence quantitative RT-PCR detection method for bluetongue virus and epidemic hemorrhagic disease virus [ J ] Chinese veterinary science, 2019,49(09):1104 and 1111 ].

4) mu.L of the total RNA of EHDV positive blood extracted in step 1) was used as a template, RNase-free water was used as a negative control, and RT-LAMP amplification was performed using the primer set II prepared in example 1.

Reaction system of RT-LAMP amplification: WarmStart LAMP 2 × Master Mix 12.5 μ L, EHDV-F31 μ L, EHDV-B31 μ L, EHDV-FIP 1 μ L, EHDV-BIP1 μ L, EHDV-LF 1 μ L, EHDV-LB 1 μ L, nucleic acid template 4 μ L, RNase-free water was added to make up to 25 μ L. The final concentration of each primer in the reaction system is as follows: 0.2. mu.M EHDV-F3, 0.2. mu.M EHDV-B3, 0.8. mu.M EHDV-FIP, 0.8. mu.M EHDV-BIP, 0.6. mu.M EHDV-LF, 0.6. mu.M EHDV-LB.

5) EHDV qRT-PCR assays were performed simultaneously with reference to literature reports (described in: establishment and application of a double fluorescence quantitative RT-PCR detection method for bluetongue virus and epidemic hemorrhagic disease virus [ J ] Chinese veterinary science, 2019,49(09):1104 and 1111 ].

6) mu.L of 21 parts of total RNA of PALV positive blood extracted in step 1) was used as a template, RNase-free water was used as a negative control, and RT-LAMP amplification was carried out using the primer set III prepared in example 1.

Reaction system of RT-LAMP amplification: WarmStart LAMP 2 × Master Mix 12.5 μ L, PALV-F31 μ L, PALV-B31 μ L, PALV-FIP 1 μ L, PALV-BIP 1 μ L, PALV-LF 1 μ L, nucleic acid template 4 μ L, RNase-free water was added to make up to 25 μ L. The final concentration of each primer in the reaction system is as follows: 0.2 μ M PALV-F3, 0.2 μ M PALV-B3, 0.8 μ M PALV-FIP, 0.8 μ M PALV-BIP, 0.6 μ M PALV-LF.

7) The PALV qRT-PCR detection was performed simultaneously according to literature report methods (described in: the establishment and application of the Palimam serogroup virus qRT-PCR detection method [ J ] the virology report 2020,36(01): 106-.

8) Determination of results

106 BTV nucleic acid positive samples are detected by RT-LAMP, the positive rate is 88.33% (106/120), 108 BTV nucleic acid positive samples are detected by qRT-PCR, the positive rate is 90.00% (108/120), and the detection result coincidence rate of the two methods is 95.0% (Table 5).

The number of EHDV nucleic acid positive samples detected by RT-LAMP is 40, the number of EHDV nucleic acid positive samples detected by qRT-PCR is 40, and the coincidence rate of the two detection methods is 100%.

The number of the positive samples of the PALV nucleic acid detected by RT-LAMP is 21, the number of the positive samples of the PALV nucleic acid detected by qRT-PCR is 21, and the coincidence rate of the two detection methods is 100%.

TABLE 5 comparison of RT-LAMP and qRT-PCR detection results of BTV nucleic acid-positive animal blood samples

3. Clinical blood sample testing

(1) Sample to be tested 1: 60 parts of EDTA anticoagulated blood sample nucleic acid is collected from sentinel cattle and sheep from Yunan Jinghong and teachers in 2020.

Clinical specimen to be examined 2: 60 parts of EDTA anticoagulated blood sample nucleic acid is collected from sentinel cattle and sheep from Yunan Jinghong and teachers in 2020.

Clinical specimen to be examined 3: 2020 total 40 parts of bovine EDTA anticoagulated blood samples collected from the areas of scenic floods and teachers in Yunnan province of the year.

(2) Primary reagents and instruments

The main reagents and apparatus were the same as in example 2.

(3) Sample detection

2) mu.L of total RNA of 60 blood samples extracted in step 1) was used as a template, and RNase-free water was used as a negative control to perform RT-LAMP amplification using the primer set I prepared in example 1.

4) mu.L of total RNA of 60 blood samples extracted in step 1) was used as a template, and RNase-free water was used as a negative control to perform RT-LAMP amplification using the primer set II prepared in example 1.

6) RT-LAMP amplification was performed using the primer set III prepared in example 1, using 4. mu.L of the total RNA of 40 blood samples extracted in step 1) as a template and RNase-free water as a negative control.

8) Determination of results

15 BTV nucleic acid positive samples are detected by RT-LAMP, 15 BTV nucleic acid positive samples are detected by qRT-PCR, and the coincidence rate of the detection results of the two methods is 100.0%;

12 parts of EHDV nucleic acid positive samples detected by RT-LAMP, 12 parts of EHDV nucleic acid positive samples detected by qRT-PCR, and the coincidence rate of the two detection methods is 100%.

8 parts of PALV nucleic acid positive samples are detected by RT-LAMP, 8 parts of PALV nucleic acid positive samples are detected by qRT-PCR, and the coincidence rate of the two detection methods is 100%.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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atacaccaag taggatgtac ytaccacata atatcttcct ggcgc 45

<210> 17

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 17

cagatctcga tcgtcctg 18

Claims

1. The primer group for detecting the bluetongue virus, the animal epidemic hemorrhagic disease virus and the Parieyamu virus is characterized in that: comprises a primer group I, a primer group II and a primer group III;

the nucleotide sequence is shown in table 1;

TABLE 1

。

2. The primer set for detecting bluetongue virus, epidemic hemorrhagic disease virus in animals and palinura virus according to claim 1, wherein: in the primer group I, the molar ratio of BTV-F3, BTV-B3, BTV-FIP, BTV-BIP, BTV-LB and BTV-LF is specifically 1: 1: 3: 3: 2: 2;

in the primer group II, the molar ratio of EHDV-F3, EHDV-B3, EHDV-FIP, EHDV-BIP, EHDV-LB and EHDV-LF can be 1: 1: 4: 4: 3: 3;

in the primer group III, the molar ratio of the PALV-F3, the PALV-B3, the PALV-FIP, the PALV-BIP and the PALV-LF is specifically 1: 1: 4: 4: 3.

3. an RT-LAMP kit comprising the primer set for detecting the bluetongue virus, the animal epidemic hemorrhagic disease virus and the Parieyamu virus according to claim 1 or 2.

4. The RT-LAMP kit of claim 3, wherein the detected sample is separately cultured virus, bovine and ovine anticoagulated blood.

5. The RT-LAMP kit of claim 3, further comprising: WarmStart LAMP 2 × Master Mix and RNase-free water.

6. The RT-LAMP kit of claim 3, wherein:

the reaction system for RT-LAMP amplification of the primer group I is as follows: WarmStart LAMP 2 × Master Mix 12.5 μ L, BTV-F31 μ L, BTV-B31 μ L, BTV-FIP 1 μ L, BTV-BIP 1 μ L, BTV-LF 1 μ L, BTV-LB 1 μ L, nucleic acid template 2 μ L, adding RNase-free water to make up to 25 μ L; the final concentration of each primer in the reaction system is as follows: 0.2. mu.M BTV-F3, 0.2. mu.M BTV-B3, 0.6. mu.M BTV-FIP, 0.6. mu.M BTV-BIP, 0.4. mu.M BTV-LF, 0.4. mu.M BTV-LB;

the reaction system for RT-LAMP amplification of the primer group II is as follows: WarmStart LAMP 2 × Master Mix 12.5 μ L, EHDV-F31 μ L, EHDV-B31 μ L, EHDV-FIP 1 μ L, EHDV-BIP1 μ L, EHDV-LF 1 μ L, EHDV-LB 1 μ L, nucleic acid template 2 μ L, adding RNase-free water to make up to 25 μ L; the final concentration of each primer in the reaction system is as follows: 0.2. mu.M EHDV-F3, 0.2. mu.M EHDV-B3, 0.8. mu.M EHDV-FIP, 0.8. mu.M EHDV-BIP, 0.6. mu.M EHDV-LF, 0.6. mu.M EHDV-LB;

the reaction system for RT-LAMP amplification of the primer group III is as follows: WarmStart LAMP 2 × Master Mix 12.5 μ L, PALV-F31 μ L, PALV-B31 μ L, PALV-FIP 1 μ L, PALV-BIP 1 μ L, PALV-LF 1 μ L, nucleic acid template 2 μ L, adding RNase-free water to make up to 25 μ L; the final concentration of each primer in the reaction system is as follows: 0.2 μ M PALV-F3, 0.2 μ M PALV-B3, 0.8 μ M PALV-FIP, 0.8 μ M PALV-BIP, 0.6 μ M PALV-LF.

7. The RT-LAMP kit of claim 3, wherein: the reaction program of RT-LAMP amplification is as follows: keeping the temperature at 64 ℃ for 45 min.

8. A method for identifying whether a virus to be tested is bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus for non-disease diagnostic and therapeutic purposes, comprising the steps of:

(2) taking the virus RNA denatured in the step (1) as a template, respectively adopting a primer group I, a primer group II and a primer group III of the primer combination to perform RT-LAMP amplification reaction, and judging according to the reaction result as follows:

if the primer group I is adopted to realize positive amplification by taking the virus RNA as a template, the virus to be detected is BTV; if the primer group II can realize positive amplification by taking the virus RNA as a template, the virus to be detected is EHDV; if the primer group III can realize positive amplification by taking the virus RNA as a template, the virus to be detected is PALV;

the positive amplification judging method comprises the following steps: after the reaction is finished, whether the amplification is positive or not is judged according to the color change of the reaction solution, if the color of the reaction solution is changed from red to yellow, the amplification is positive, and if the color of the reaction solution is kept unchanged from red, the amplification is negative.

9. A method for identifying whether a test sample contains BTV and/or EHDV and/or PALV for non-disease diagnostic and therapeutic purposes, comprising the steps of:

if the primer group I is adopted, the positive amplification with the total RNA of the sample to be detected as a template can be realized, and the sample to be detected contains BTV; if the primer group II is adopted, the positive amplification with the total RNA of the sample to be detected as a template can be realized, and the sample to be detected contains EHDV; if the primer group III is adopted, the positive amplification with the total RNA of the sample to be detected as a template can be realized, and the sample to be detected contains PALV; if the primer group I, the primer group II and the primer group III can not realize positive amplification by taking the total RNA of the sample to be detected as a template, the sample to be detected does not contain BTV, EHDV and PALV;