Quadruple fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for detecting ruminant arthroviruses as well as use method and application

Abstract

The invention discloses a quadruple fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for detecting ruminant arthroviruses as well as a use method and application thereof, and belongs to the technical field of molecular biology. In order to solve the problem that a rapid, sensitive and specific detection method capable of simultaneously detecting BTV, EHDV, AKAV and CHUV viruses lacks in the prior art, the invention provides a quadruple fluorescent quantitative RT-PCR detection kit for detecting ruminant arthroviruses, the kit comprises a primer pair and a probe combination, and the quadruple fluorescent quantitative RT-PCR detection method. The detection method has good reaction specificity, sensitivity and repeatability, and the quadruple fluorescent quantitative RT-PCR detection method has high positive rate accuracy on AKAV, BTV, EHDV and CHUV detection results, and can be applied to conventional detection of clinical samples and laboratory samples and daily monitoring of ruminant groups.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a detection kit for detecting ruminant insect-borne viruses and a use method and application thereof. Background Art

[0002] Bluetongue virus (BTV) is a member of the Obivirus genus of the Reoviridae family. The virus particle is about 80nm in diameter and has icosahedral symmetry. Its genome consists of 10 segments of double-stranded RNA (dsRNA). BTV is transmitted by vector insects of the genus Culicoides and mainly infects ruminants such as sheep, goats, and cattle, of which sheep are the most susceptible. Bluetongue is an animal disease that is legally notifiable to the World Organization for Animal Health (WOAH). The main clinical manifestations include fever, salivation, cyanosis of the tongue, erosion or ulceration of the oral mucosa, and shedding of hoof shells in severe cases. Infection with some serotypes can cause abortion in pregnant females, the birth of mummified fetuses, and reproductive disorders. At present, at least 29 different serotypes of BTV (BTV-1 to BTV-29) have been isolated and identified worldwide, as well as several BTV isolates whose serotypes have not yet been finally confirmed, preliminarily identified as BTV-30 to BTV-36. my country has detected many different serotypes, including BTV-1, -2, -3, -4, -5, -7, -9, -12, -14, -15, -16, -20, and -21.

[0003] Epizootic haemorrhagic disease virus (EHDV) belongs to the genus Orbivirus of the family Reoviridae. The virus particles are icosahedral symmetrical, with a diameter of about 80nm and a double-layer capsid structure. Its genome consists of 10 segments of dsRNA. EHDV is transmitted between animals by blood-sucking insects such as Culicoides as a medium, and mainly infects ruminants such as deer, cattle, and sheep. Among them, white-tailed deer is the species most seriously affected by EHDV. Epizootic haemorrhagic disease is a transboundary animal disease that is notifiable by WOAH. The main clinical symptoms are high fever, anorexia, weakness, swollen and cyanotic tongue, respiratory distress, edema of the head and neck, congestion of the nasal mucosa, nasal erosion, etc. Infection with some serotypes of the virus can cause reproductive disorders such as abortion, stillbirth, and fetal brain malformation in pregnant animals. At present, 10 different serotypes of EHDV have been discovered in the world, including EHDV-1, -2, -4 to -11. Among them, my country has detected multiple serotypes of EHDV-1, -2, -5, -6, -7, -8, -10 and -11.

[0004] Akabane virus (AKAV) is a member of the Simbu virus serogroup of the Bunyaviridae family and the genus Orthobunyavirus. The virus particle is about 80-120nm in diameter and is spherical with an envelope. The viral genome is a single-stranded negative-strand segmented RNA with three segments. AKAV is transmitted between animals through the bites of blood-sucking insects such as Culicoides and mosquitoes. Susceptible animals are domestic ruminants such as cattle, sheep, and goats. There are also records of AKAV positive detection in animals such as wildebeest, antelope, buffalo, and camels. Akabane disease is a legally notifiable disease of WOAH. AKAV infection in adult cattle and sheep is mostly latent. The main clinical symptoms of the virus infecting the fetus are abortion, stillbirth, fetal body abnormalities or deformities (such as newborn arthrogryposis and hydrocephalus syndrome), etc., causing reproductive disorders. It is currently believed that there is only one serotype of AKAV and four gene groups (Asian group, Australian group, other groups and the fourth gene group). There are records of positive AKAV tests in ruminant populations in Shandong, Shanghai, Beijing, Hunan, Guangxi, Zhejiang, Fujian, Gansu, Hebei, Inner Mongolia, Jilin and other regions in my country.

[0005] Chuzan virus (CHUV) is a member of the Palyamserogroup of the genus Orbivirus of the family Reoviridae. The virus particle is about 50nm, round and non-enveloped, and the viral genome consists of 10 segments of dsRNA. CHUV is transmitted between ruminants such as cattle and sheep by bites of Culicoides as a transmission medium and storage host, and cattle are the most susceptible animals. CHUV infection in adult cattle is mostly latent infection without obvious clinical symptoms; infection in pregnant cows mainly manifests reproductive disorders such as miscarriage, stillbirth, and birth of malformed fetuses; infection in calves may manifest as hydrocephalus and cerebellar hypoplasia. In 2013, CHUV was isolated in Guangxi for the first time in my country, and positive CHUV detection records have been found in Inner Mongolia, Xinjiang, Shandong, Jiangsu, Hubei, Guangxi, Yunnan, Taiwan and other places.

[0006] Ruminants such as cattle and sheep are an important part of my country's animal husbandry. Epidemiological surveys show that the infection rates of BTV, EHDV, AKAV, and CHUV in my country's ruminant populations are high, and multiple strains of BTV and EHDV, as well as AKAV and CHUV strains of different serotypes have been isolated. First, there are many types of vector insects with transmission capabilities in China, and the environment and climate in the south are suitable for the reproduction of vector insects, which is conducive to the rapid spread and spread of viral infections. Secondly, in recent years, the breeding volume of cattle, sheep, deer, etc. has increased rapidly and continuously, the susceptible animal population has continued to expand, and international agricultural and animal husbandry and livestock product trade has become increasingly frequent. The above factors are highly alarming, and insect-borne infectious diseases have posed a serious threat to my country's ruminant breeding industry. In addition, BTV, EHDV, AKAV, and CHUV have the same transmission vectors and susceptible animal populations, and there is overlap in the epidemic areas, which poses a risk of mixed infection.

[0007] In the prior art, most of the detection methods for AKAV, BTV, CHUV, and EHDV are single detection, which is difficult to meet the needs of detecting multiple diseases at the same time; therefore, technical personnel in this field are eager to develop a rapid, sensitive, and specific detection method that can simultaneously detect the above four viruses, which can be applied to daily monitoring and early differential diagnosis of diseases. Summary of the invention

[0008] The present invention solves the problem of the lack of a rapid, sensitive, specific detection method capable of simultaneously detecting BTV, EHDV, AKAV and CHUV viruses in the prior art, and provides a quadruple fluorescent quantitative RT-PCR detection kit for detecting ruminant insect-borne viruses, as well as a use method and application thereof.

[0009] One of the purposes of the present invention is to provide a quadruple fluorescent quantitative RT-PCR detection kit for detecting ruminant insect-borne viruses, wherein the quadruple fluorescent quantitative RT-PCR detection kit comprises a primer pair and a probe combination, including the following components:

[0010] The specific primer pair and probe nucleotide sequences for detecting AKAV are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively;

[0011] The specific primer pair and probe nucleotide sequences for detecting BTV are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively;

[0012] The specific primer pair and probe nucleotide sequences for detecting CHUV are shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively;

[0013] The specific primer pair and probe nucleotide sequences for detecting EHDV are shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 respectively.

[0014] In a preferred embodiment of the present invention, the specific primer pairs and probes of AKAV, BTV, CHUV and EHDV use the conservative sequence fragment of AKAV genome segment S, the conservative sequence fragment of BTV genome segment S10, the conservative sequence fragment of CHUV genome segment S3 and the conservative sequence fragment of EHDV genome segment S9 as templates, respectively.

[0015] In a preferred embodiment of the present invention, the quadruple fluorescent quantitative RT-PCR detection kit also includes: 2×One Step U+Mix 10.0μL, One Step U+Enzyme Mix 1.0μL, 50×ROX Reference Dye II 0.4μL, nucleic acid template 1.0μL, 10μM primer pair 0.4-1.0μL, 10μM probe 0.4-0.6μL, and the total reaction volume is 20.0μL.

[0016] In a preferred embodiment of the present invention, the amount of the primer pair added is specifically:

[0017] The amount of primer pair added for detecting AKAV was 0.6 μL, and the amount of probe added was 0.6 μL;

[0018] The amount of primer pair added for detecting BTV was 0.8 μL, and the amount of probe added was 0.8 μL;

[0019] The amount of primer pair added for detecting CHUV was 1.0 μL, and the amount of probe added was 0.6 μL;

[0020] The amount of the primer pair added for detecting EHDV was 0.8 μL, and the amount of the probe added was 0.8 μL.

[0021] The second object of the present invention is to provide a method for using the above-mentioned quadruple fluorescent quantitative RT-PCR detection kit for non-diagnostic purposes, and the method comprises the following steps: using the total nucleic acid extracted from the object to be detected as a template, using the four specific primer pairs and probe sequences in the quadruple fluorescent quantitative RT-PCR detection kit, and the quadruple fluorescent quantitative RT-PCR reaction system to perform a quadruple fluorescent quantitative RT-PCR reaction, and finally interpreting the qualitative and quantitative results.

[0022] In a preferred embodiment of the present invention, the procedure of the quadruple fluorescent quantitative RT-PCR reaction in the method of use is: reverse transcription at 55°C for 15 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing and extension at 56°C for 30 s; a total of 45 cycles.

[0023] In a preferred embodiment of the present invention, the qualitative result interpretation method is as follows: when the quality of the quadruple fluorescent quantitative RT-PCR test is under control, if the amplification curve shows an S-type or exponential growth, and the CT value is less than 39, the test result is determined to be positive; if the CT value is greater than 42, the test result is determined to be negative; if the amplification curve shows an S-type or exponential growth, and the CT value is greater than or equal to 39 and less than or equal to 42, the test result is determined to be uncertain, and the nucleic acid needs to be re-extracted for re-examination; if the amplification curve shows an S-type or exponential growth in the re-examination, and the CT value is less than 42, the test result is determined to be positive, otherwise it is negative.

[0024] In a preferred embodiment of the present invention, the quantitative result is interpreted as follows: the plasmid standard is diluted in a gradient manner, converted into the copy number according to the molecular weight of the target gene, a standard curve is drawn with the Ct value after amplification as the ordinate and the logarithm of the copy number as the abscissa, the Ct value of the object to be detected is substituted into the standard curve, and the copy number of AKAV S, BTV S10, CHUV S3, and EHDV S9 genes is calculated for quantitative detection.

[0025] The third object of the present invention is to provide the use of the above-mentioned quadruple fluorescent quantitative RT-PCR detection kit in detecting ruminant arboviruses, wherein the ruminant arboviruses are AKAV, BTV, CHUV and EHDV.

[0026] The beneficial effects of the present invention are as follows: the present invention provides a quadruple fluorescent quantitative RT-PCR detection kit for detecting ruminant arboviruses, the kit comprising a primer pair and a probe combination (nucleotide sequence is shown in SEQ ID NO.1-12), and a quadruple fluorescent quantitative RT-PCR detection reaction system and a reaction procedure; the BTV primers and probes can specifically detect at least 33 different serotypes of BTV, including BTV-1 to BTV-29, BTV-31 to BTV-34; the EHDV primers and probes can specifically detect at least 8 different serotypes of EHDV, including EHDV-1, EHDV-2, EHDV-4 to EHDV-8, and EHDV-10.

[0027] The quadruple fluorescent quantitative RT-PCR detection kit provided by the present invention successfully amplifies only the four target genes of AKAV, BTV, CHUV, and EHDV without cross-reaction; the minimum detection limit of the quadruple fluorescent quantitative RT-PCR detection kit for AKAV, BTV, CHUV, and EHDV is 100 copies / reaction; the results of repeated intra-group and inter-group detection of the quadruple fluorescent quantitative RT-PCR detection kit of the present invention show that the intra-group coefficient of variation is between 0.10% and 0.92%, the inter-group coefficient of variation is between 1.10% and 2.53%, and the coefficient of variation of repeated intra-group and inter-group detection is below 3%. In summary, the quadruple fluorescent quantitative RT-PCR detection kit provided by the present invention and the method of use thereof have good reactivity, specificity, sensitivity, and repeatability.

[0028] In the quadruple fluorescent quantitative RT-PCR detection kit provided by the present invention, the sample detection positive rates of AKAV, BTV, CHUV and EHDV are consistent with the published single fluorescent quantitative RT-PCR detection results; it can be seen that the quadruple fluorescent quantitative RT-PCR detection kit provided by the present invention can simultaneously detect the above four viruses in the same reaction tube, can be used for routine detection of clinical samples and laboratory samples and daily monitoring of ruminant groups, provides a faster, more convenient and more efficient detection method for ruminant insect-borne viral infectious diseases, and provides a new technology for the detection of AKAV, BTV, CHUV and EHDV. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Figure 2 is a graph showing the optimization of primer and probe concentrations for a single fluorescent quantitative RT-PCR reaction in Example 2; A is a graph showing AKAV detection, B is a graph showing BTV detection, C is a graph showing CHUV detection, and D is a graph showing EHDV detection;

[0030] Figure 2 It is the optimization diagram of annealing temperature of single fluorescence quantitative RT-PCR reaction in Example 2; A is the detection diagram of AKAV, B is the detection diagram of BTV, C is the detection diagram of CHUV, and D is the detection diagram of EHDV;

[0031] Figure 3 Figure 2 is the amplification curve and standard curve of the AKAV single fluorescence quantitative RT-PCR detection in Example 2; A is the amplification curve, and B is the standard curve;

[0032] Figure 4 The amplification curve and standard curve of the BTV single fluorescence quantitative RT-PCR detection in Example 2; A is the amplification curve, and B is the standard curve;

[0033] Figure 5The amplification curve and standard curve of CHUV single fluorescence quantitative RT-PCR detection in Example 2; A is the amplification curve, and B is the standard curve;

[0034] Figure 6 The amplification curve and standard curve of the single fluorescence quantitative RT-PCR detection of EHDV in Example 2; A is the amplification curve, and B is the standard curve;

[0035] Figure 7 Specific detection diagrams of single fluorescent quantitative RT-PCR in Example 2; A is the AKAV detection diagram, B is the BTV detection diagram, C is the CHUV detection diagram, and D is the EHDV detection diagram;

[0036] Figure 8 Figure 2 is a sensitivity detection diagram of the single fluorescent quantitative RT-PCR in Example 2; A is the AKAV detection diagram, B is the BTV detection diagram, C is the CHUV detection diagram, and D is the EHDV detection diagram;

[0037] Fig. 9 The amplification curve and standard curve of the quadruple fluorescent quantitative RT-PCR detection in Example 3; A is the amplification curve, and B is the standard curve;

[0038] Fig.10 Specific detection diagrams of quadruple fluorescent quantitative RT-PCR in Example 3, A is a detection diagram of a mixed nucleic acid sample of four viruses, and B is a detection diagram of a single viral nucleic acid sample;

[0039] Fig.11 : Figure 3 is the sensitivity detection graph of the quadruple fluorescent quantitative RT-PCR in Example 3; A is the sensitivity detection summary result graph; B is the AKAV detection graph, C is the BTV detection graph, D is the CHUV detection graph, and E is the EHDV detection graph. DETAILED DESCRIPTION

[0040] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0041] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification. The test methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0042] Materials and instruments used in the following examples:

[0043] Virus samples: Akavanese disease virus (AKAV), bluetongue virus (BTV), Chungshan disease virus (CHUV), epidemic hemorrhagic disease virus (EHDV), peste des petits veterinary virus (PPRV), goat pox virus (GPV), bovine respiratory syncytial virus (BRSV), bovine rotavirus (BRV), bovine parainfluenza virus type 3 (BPIV3) and bovine infectious rhinotracheitis virus (IBRV) have all been inactivated. All of the above viruses were donated by the Cattle and Sheep Infectious Diseases Innovation Team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0044] Plasmid samples: The recombinant plasmids of AKAV-S gene, BTV-S10 gene, CHUV-S3 gene, and EHDV-S9 gene were named pCI-AKAV-S plasmid, pCI-BTV-S10 plasmid, pCI-CHUV-S3 plasmid, and pCI-EHDV-S9 plasmid, respectively. The above plasmids were donated by the cattle and sheep infectious disease innovation team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The tandem gene fragment containing AKAV-S gene, BTV-S10 gene, CHUV-S3 gene, and EHDV-S9 gene was synthesized by Jilin Kumei Biotechnology Co., Ltd. and inserted into the pUC57 cloning vector to form a tandem standard plasmid named pUC57-AKAV-BTV-CHUV-EHDV plasmid.

[0045] Reagents: Trizol (ThermoFisher Scientific), endotoxin-free plasmid miniprep kit (TIANGEN), AccurSTART U+One Step RT-qPCR Probe Kit (Vazyme), EASY Dilution (TaKaRa), DH5α competent cells (Shanghai Sangon Biotech Co., Ltd.).

[0046] Instruments: Electric constant temperature water bath (Shanghai Yiheng Technology Co., Ltd.), Centrifuge 5418 centrifuge (Eppendorf), QuantStudio 5 real-time fluorescence quantitative PCR instrument (ABI), handheld centrifuge (SCILOGEX), adjustable mixer (Beyotime).

[0047] Example 1: Preparation of primers, probes, nucleic acid samples and standards

[0048] (1) Design of primers and probes

[0049] In this example, the genome sequences of AKAV, different serotypes of BTV, CHUV, and different serotypes of EHDV were downloaded from the Genbank database, and after comparison and analysis, the AKAV-S segment, BTV-S10 segment, CHUV-S3 segment, and EHDV-S9 segment were selected as detection target sequences, and primers and fluorescent probes were designed; the nucleotide sequences of the primers and probes are shown in Table 1.

[0050] Table 1

[0051]

[0052]

[0053] Note: Y represents the degenerate base C+T, R represents the degenerate base A+G, K represents the degenerate base G+T, W represents the degenerate base A+T, M represents the degenerate base A+C, and N represents the degenerate base A+T+C+G.

[0054] (2) Preparation of nucleic acid samples and standards

[0055] In this example, Trizol reagent was used to extract AKAV, BTV-1 / 12 / 15 / 16 / 20, CHUV, EHDV-2 / 5 / 7 and PPRV, GPV, BRSV, BRV, BPIV3, and IBRV genomic RNA according to its instructions, and the RNA was placed in a -80°C refrigerator for later use.

[0056] The pCI-AKAV-S plasmid, pCI-BTV-S10 plasmid, pCI-CHUV-S3 plasmid, pCI-EHDV-S9 plasmid and pUC57-AKAV-BTV-CHUV-EHDV plasmid were extracted using an endotoxin-free plasmid mini-extraction kit, and the concentrations of the above plasmids were measured using an ultra-micro-UV spectrophotometer to obtain the copy numbers of the above plasmids. Then, the above plasmids were diluted to 1×10 9 -1×10 0 copies / μL and stored in a -20℃ refrigerator for later use.

[0057] Example 2: Construction and verification of a single fluorescent quantitative RT-PCR detection reaction system

[0058] (1) Construction of single fluorescence quantitative RT-PCR detection reaction system

[0059] In this example, the matrix method was used to optimize the amounts of primers and probes and the reaction temperature in the single fluorescent quantitative RT-PCR reaction system of AKAV, BTV, CHUV and EHDV.

[0060] The reaction procedure of fluorescence quantitative RT-PCR detection was as follows: reverse transcription at 55°C for 15 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, and a total of 45 cycles of denaturation and extension.

[0061] The fluorescence quantitative RT-PCR detection reaction system was 2×One Step U+Mix 10.0μL, One Step U+Enzyme Mix 1.0μL, 50×ROX Reference Dye II 0.4μL, and nucleic acid template 1.0μL, with a total reaction volume of 20.0μL; the primer dosage was optimized in the range of 0.4-1.2μL (0.4, 0.6, 0.8, 1.0, 1.2μL), and the probe dosage was optimized in the range of 0.2-0.8μL (0.2, 0.4, 0.6, 0.8μL). The concentrations of primers and probes were both 10μM. The optimization results of primer and probe concentrations for single fluorescence quantitative RT-PCR reactions are shown in Figure 2. Figure 1 shown.

[0062] In the fluorescence quantitative RT-PCR detection reaction system, the AKAV S F1-1 and AKAV S R1 primer pair were selected with a dosage of 0.6 μL, and the AKAV S P1 probe was selected with a dosage of 0.6 μL; the BTV S10 F2-3 and BTV S10 R2-3 primer pair were selected with a dosage of 0.8 μL, and the BTV S10 P2 probe was selected with a dosage of 0.8 μL; the CHUV S3 F2-1 and CHUV S3 R2 primer pair were selected with a dosage of 1.0 μL, and the CHUV S3 P2 probe was selected with a dosage of 0.6 μL; the EHDV S9 F2-2 and EHDV S9 R2-1 primer pair were selected with a dosage of 0.8 μL, and the EHDV S9 P2 probe was selected with a dosage of 0.8 μL.

[0063] On the basis of the primer pairs and probe amounts of the above-mentioned single fluorescence quantitative RT-PCR reactions for AKAV, BTV, CHUV, and EHDV, and other reaction procedures remaining unchanged, the annealing temperature of the fluorescence quantitative RT-PCR reaction was adjusted within the range of 52°C-62°C (52°C, 54°C, 56°C, 58°C, 60°C, and 62°C). Figure 2 As shown, the optimal annealing temperature for single fluorescence quantitative RT-PCR reactions of AKAV, BTV, CHUV, and EHDV was 56°C.

[0064] (2) Establishment of single fluorescence quantitative RT-PCR detection standard curve

[0065] In this example, the pCI-AKAV-S plasmid, pCI-BTV-S10 plasmid, pCI-CHUV-S3 plasmid, and pCI-EHDV-S9 plasmid obtained in Example 1 were diluted 10 times, and 1×10 8 copies / μL-1×10 1 8 different concentration gradients were used as templates for single fluorescence quantitative RT-PCR amplification reaction. Standard curves were prepared respectively, among which the single fluorescence quantitative standard curve of AKAV was y=-3.197log(X)+38.345, R 2 =0.998, Eff% = 105.496 (e.g. Figure 3 The single fluorescence quantitative standard curve of BTV is y=-3.314log(X)+38.800, R 2 =0.998, Eff% = 100.324 (such as Figure 4 As shown); CHUV single fluorescence quantitative standard curve is y = -3.293log (X) + 39.507, R 2 =1.000, Eff%=101.213 (e.g. Figure 5 EHDV single fluorescence quantitative standard curve is y = -3.297log (X) + 39.446, R 2 =0.999, Eff% = 101.059 (such as Figure 6 shown).

[0066] It can be seen that the single fluorescent quantitative RT-PCR reaction system of AKAV, BTV, CHUV and EHDV provided by the present invention can be used for the detection of AKAV, BTV, CHUV and EHDV at 1×10 8 copies / μL-1×10 1 There was a good linear relationship in the 8 different concentration gradient ranges.

[0067] (3) Specificity verification of single fluorescence quantitative RT-PCR detection

[0068] The AKAV, BTV-1 / 12 / 15 / 16 / 20, CHUV, and EHDV-2 / 5 / 7 genomes were used as positive nucleic acid samples, and the PPRV, GPV, BRSV, BRV, BPIV3, and IBRV genome RNA obtained in Example 1 were used as control samples. The primers and probes of the four pathogens AKAV, BTV, CHUV, and EHDV were used to verify the specificity of single fluorescence quantitative RT-PCR detection of the above samples.

[0069] The results are as follows Figure 7As shown, only four target genes, AKAV, BTV, CHUV and EHDV, were successfully amplified without cross-reaction, which proved that the corresponding primers and probes provided by the present invention have good reactivity and specificity.

[0070] (4) Sensitivity verification of single fluorescence quantitative RT-PCR detection

[0071] In this example, the pCI-AKAV-S plasmid, pCI-BTV-S10 plasmid, pCI-CHUV-S3 plasmid, and pCI-EHDV-S9 plasmid obtained in Example 1 were diluted 10 times, and 1×10 9 copies / μL-1×10 0 10 copies / μL of plasmids with different concentration gradients were used as templates for single fluorescence quantitative RT-PCR amplification reaction. 1 copies / μL plasmid and 1×10 0 The PCR products were tested 28 times with the 100 copies / μL plasmid as template, and a CT value of ≤39 was considered positive. The positive detection rate of the 28 repeated tests was calculated. If the positive detection rate was ≥75%, the concentration was determined to be detectable.

[0072] Sensitivity test results Figure 8 As shown in Table 2 , the minimum detection limit of the single fluorescence quantitative RT-PCR detection method for AKAV, BTV, CHUV, and EHDV was 10 copies / reaction.

[0073] Table 2

[0074]

[0075] (5) Repeatability verification of single fluorescence quantitative RT-PCR detection

[0076] In this example, 1×10 6 copies / μL-1×10 3 The pCI-AKAV-S plasmid, pCI-BTV-S10 plasmid, pCI-CHUV-S3 plasmid, and pCI-EHDV-S9 plasmid obtained in (1) with a total of 4 dilution concentrations of 100 copies / μL were subjected to single fluorescence quantitative RT-PCR repeatability detection, and repeated detection within and between groups was performed; 3 replicates of each dilution concentration of the plasmid in a group were performed as an within-group test; the above experiment was repeated 3 times as an between-group test.

[0077] The repeatability verification results are shown in Table 3. The intra-group coefficient of variation was between 0.17% and 1.69%, the inter-group coefficient of variation was between 0.36% and 2.53%, and the coefficients of variation of repeated detection within and between groups were both below 3%, proving that the single fluorescence quantitative RT-PCR detection method provided by the present invention has good repeatability.

[0078] Table 3

[0079]

[0080]

[0081] Example 3: Construction and verification of the reaction system and reaction procedure in the quadruple fluorescence quantitative RT-PCR detection kit

[0082] (1) Construction of the reaction system and reaction procedure in the quadruple fluorescence quantitative RT-PCR detection kit

[0083] The pUC57-AKAV-BTV-CHUV-EHDV plasmid obtained in Example 1 was diluted 10 times. The quadruple fluorescence quantitative RT-PCR detection reaction system is shown in Table 4, wherein the primer and probe concentrations are both 10 μM, and the quadruple fluorescence quantitative RT-PCR reaction procedure is: reverse transcription at 55°C for 15 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing and extension at 56°C for 30 s; a total of 45 cycles.

[0084] Table 4

[0085]

[0086]

[0087] (2) Establishment of standard curve for quadruple fluorescence quantitative RT-PCR detection kit

[0088] Select 1×10 9 copies / μL-1×10 2 The plasmid obtained in (1) was used as a template for a quadruple fluorescence quantitative RT-PCR reaction. The results are shown in Fig. 9 shown.

[0089] The standard curve of AKAV multiple fluorescence quantitative analysis is y = -3.306log(X) + 39.063, R 2 =0.999, Eff%=100.682;

[0090] The BTV multiple fluorescence quantitative standard curve is y = -3.374log (X) + 40.128, R 2=0.999, Eff% = 97.884;

[0091] The CHUV multiple fluorescence quantitative standard curve is y = -3.403log (X) + 39.892, R 2 =0.996, Eff% = 96.724;

[0092] The EHDV multiple fluorescence quantitative standard curve is y = -3.286log (X) + 39.410, R 2 =0.999,Eff%=101.513.

[0093] The results show that the quadruple fluorescence quantitative RT-PCR detection kit provided by the present invention has a good linear relationship within the dilution range.

[0094] (3) Specificity verification of the quadruple fluorescence quantitative RT-PCR detection kit

[0095] The genomes of AKAV, BTV-1 / 12 / 15 / 16 / 20, CHUV, and EHDV-2 / 5 / 7 were used as positive nucleic acid samples, and the genomes of PPRV, GPV, BRSV, BRV, BPIV3, and IBRV were used as controls. The primers and probes of the four pathogens were used to verify the specificity of the quadruple fluorescence quantitative RT-PCR detection kit.

[0096] The results are as follows Fig.10 As shown, only four target genes, AKAV, BTV, CHUV and EHDV, were successfully amplified without cross-reaction, which proves that the primers and probes provided by the present invention have good reactivity and specificity.

[0097] (4) Sensitivity verification of the quadruple fluorescence quantitative RT-PCR detection kit

[0098] In this example, 1×10 9 copies / μL-1×10 0 The pUC57-AKAV-BTV-CHUV-EHDV plasmid obtained in 10 different concentration gradients (1) was used as a template for quadruple fluorescence quantitative RT-PCR amplification reaction. 2 copies / μL plasmid and 1×10 1 The PCR products were tested 28 times with the 100 copies / μL plasmid as template, and a CT value of ≤39 was considered positive. The positive detection rate of the 28 repeated tests was calculated. If the positive detection rate was ≥75%, the concentration was determined to be detectable.

[0099] Sensitivity test results Fig.11As shown in Table 5, the detection results of the quadruple fluorescence quantitative RT-PCR detection kit provided by the present invention for the pUC57-AKAV-BTV-CHUV-EHDV plasmid with a concentration of 100 copies / μL showed that the positive detection rates of the four target genes AKAV, BTV, CHUV and EHDV were all 100%; the detection results of the pUC57-AKAV-BTV-CHUV-EHDV plasmid with a concentration of 10 copies / reaction showed that the positive detection rates of the four target genes were AKAV 57%, BTV 46%, CHUV 57%, and EHDV 57%, respectively.

[0100] Therefore, in the detection reaction of the quadruple fluorescent quantitative RT-PCR detection kit provided by the present invention, the minimum detection limit of AKAV, BTV, CHUV and EHDV is 100 copies / reaction.

[0101] Table 5

[0102]

[0103] (5) Repeatability verification of quadruple fluorescence quantitative RT-PCR detection kit

[0104] In this example, 1×10 6 copies / μL-1×10 3 The pUC57-AKAV-BTV-CHUV-EHDV plasmid obtained in (1) with 4 dilution concentrations of 100 copies / μL was subjected to multiple fluorescence quantitative RT-PCR repeatability detection, and repeated detection within and between groups was performed; 3 replicates of the plasmid at each dilution concentration in a group were performed as an within-group test; the above experiment was repeated 3 times as an between-group test.

[0105] The repeatability verification results are shown in Table 6. The intra-group coefficient of variation is between 0.10% and 0.92%, the inter-group coefficient of variation is between 1.10% and 2.53%, and the coefficients of variation of intra-group and inter-group repeated detection are both below 3%, proving that the quadruple fluorescent quantitative RT-PCR detection kit provided by the present invention has good repeatability.

[0106] Table 6

[0107]

[0108]

[0109] (6) Sample testing of the quadruple fluorescence quantitative RT-PCR detection kit:

[0110] In this example, the feasibility of the quadruple fluorescence quantitative RT-PCR detection kit was verified, and 138 samples (including clinical cattle and sheep blood samples and laboratory samples) were detected using the quadruple fluorescence quantitative RT-PCR detection kit. At the same time, the published single fluorescence quantitative RT-PCR detection methods for AKAV, BTV, CHUV and EHDV were used to detect the above samples respectively, and the detection results were compared; among them, the published single fluorescence quantitative RT-PCR detection method for BTV was based on GB / T18636-2017 bluetongue disease diagnosis technology, the published single fluorescence quantitative RT-PCR detection method for AKAV was based on SN_T 3991-2014 Akabane virus real-time fluorescence RT-PCR detection method, and the published single fluorescence quantitative RT-PCR detection methods for EHDV and CHUV were based on research papers (Maan NS, Maan S, Potgieter AC, Wright IM, Belaganahalli M, Mertens PPC. Development of Real-Time RT-PCR Assays for Detection and Typing of Epizootic Haemorrhagic Disease Virus (Establishment of Real-Time RT-PCR Detection and Typing of Epizootic Haemorrhagic Disease Virus). Transbound Emerg Dis.2017Aug;64(4):1120-1132; Yang Zhenxing, Zhu Pei, Xie Jiarui, Xiao Lei, Yang Heng, Liao Defang, Li Huachun, Zhu Jianbo. Establishment and application of qRT-PCR and RT-PCR detection methods for Zhongshan disease virus [J]. Chinese Journal of Preventive Veterinary Medicine, 2021, 43(12):1275-1281.).

[0111] The results are shown in Table 7. The quadruple fluorescence quantitative RT-PCR detection kit provided by the present invention was used for sample detection. The positive rate of AKAV was 5.80% (8 / 138), which was consistent with the detection result of the real-time fluorescence RT-PCR detection method of Akabane virus in SN_T 3991-2014; the positive rate of BTV was 15.94% (22 / 138), which was consistent with the detection result of the single fluorescence quantitative RT-PCR detection method in the bluetongue diagnosis technology of GB / T 18636-2017; the positive rate of CHUV was 3.62% (5 / 138), which was consistent with the detection result of the detection method in the research paper published on CHUV; the positive rate of EHDV was 9.42% (13 / 138), which was consistent with the detection result of the detection method in the research paper published on EHDV.

[0112] This shows that the detection results of the quadruple fluorescent quantitative RT-PCR detection kit provided by the present invention have a high consistency with the detection results of the four published single fluorescent quantitative RT-PCR detection methods for AKAV, BTV, CHUV, and EHDV. Therefore, the quadruple fluorescent quantitative RT-PCR detection kit provided by the present invention can simultaneously detect the above four viruses in the same reaction tube, and can be used for routine detection of clinical samples and laboratory samples and daily monitoring of ruminant groups, providing a faster, more convenient and more efficient detection method for ruminant insect-borne viral infectious diseases.

[0113] Table 7

[0114]

[0115] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. A quadruple fluorescent quantitative RT-PCR detection kit for detecting ruminant insect-borne viruses, characterized in that: The ruminant arboviruses are AKAV, BTV, CHUV and EHDV, and the quadruple fluorescent quantitative RT-PCR detection kit comprises a primer pair and a probe combination, including the following components: The specific primer pair and probe nucleotide sequences for detecting AKAV are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; The specific primer pair and probe nucleotide sequences for detecting BTV are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively; The specific primer pair and probe nucleotide sequences for detecting CHUV are shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively; The specific primer pair and probe nucleotide sequences for detecting EHDV are shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 respectively.

2. The quadruple fluorescent quantitative RT-PCR detection kit according to claim 1, characterized in that: The specific primer pairs and probes of AKAV, BTV, CHUV and EHDV respectively use the conservative sequence fragment of AKAV genome segment S, the conservative sequence fragment of BTV genome segment S10, the conservative sequence fragment of CHUV genome segment S3 and the conservative sequence fragment of EHDV genome segment S9 as templates.

3. The quadruple fluorescence quantitative RT-PCR detection kit according to claim 1, characterized in that: The quadruple fluorescent quantitative RT-PCR detection kit also includes: 2×One Step U+Mix 10.0μL, One Step U+Enzyme Mix 1.0μL, 50×ROX Reference Dye II 0.4μL, nucleic acid template 1.0μL, 10μM primer pair 0.4-1.0μL, 10μM probe 0.4-0.6μL, and the total reaction volume is 20.0μL.

4. The quadruple fluorescent quantitative RT-PCR detection kit according to claim 3, characterized in that: The amount of primer pair added is specifically: The amount of primer pair added for detecting AKAV was 0.6 μL, and the amount of probe added was 0.6 μL; The amount of primer pair added for detecting BTV was 0.8 μL, and the amount of probe added was 0.8 μL; The amount of primer pair added for detecting CHUV was 1.0 μL, and the amount of probe added was 0.6 μL; The amount of the primer pair added for detecting EHDV was 0.8 μL, and the amount of the probe added was 0.8 μL.

5. A method for using the quadruple fluorescent quantitative RT-PCR detection kit according to any one of claims 1 to 4 for non-diagnostic purposes, characterized in that: The method of use comprises the following steps: using the total nucleic acid extracted from the object to be detected as a template, using the four specific primer pairs and probe sequences in the quadruple fluorescent quantitative RT-PCR detection kit, and the quadruple fluorescent quantitative RT-PCR reaction system to perform a quadruple fluorescent quantitative RT-PCR reaction, and finally interpreting the qualitative results and the quantitative results.

6. The method of use according to claim 5, characterized in that: The procedure of the quadruple fluorescent quantitative RT-PCR reaction in the method is: reverse transcription at 55°C for 15 minutes; pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 10 seconds, annealing and extension at 56°C for 30 seconds; a total of 45 cycles.

7. The method of use according to claim 5, characterized in that: The qualitative result interpretation method is as follows: when the detection quality of the quadruple fluorescent quantitative RT-PCR is under control, if the amplification curve shows an S-shaped or exponential growth, and the CT value is less than 39, the detection result is determined to be positive; if the CT value is greater than 42, the detection result is determined to be negative; If the amplification curve shows an S-shaped or exponential growth, and the CT value is greater than or equal to 39 and less than or equal to 42, the test result is determined to be uncertain and the nucleic acid needs to be re-extracted for re-testing; If the amplification curve in the retest shows an S-shaped or exponential growth, and the CT value is less than 42, the test result is judged to be positive, otherwise it is negative.

8. The method of use according to claim 5, characterized in that: The quantitative result interpretation method is as follows: the plasmid standard is diluted in a gradient manner, and the molecular weight of the target gene is converted into the copy number. A standard curve is drawn with the Ct value after amplification as the ordinate and the logarithm of the copy number as the abscissa. The Ct value of the object to be detected is substituted into the standard curve, and the copy number of AKAV S, BTV S10, CHUVS3, and EHDV S9 genes is calculated for quantitative detection.

9. Use of the quadruple fluorescent quantitative RT-PCR detection kit according to any one of claims 1 to 4 in detecting ruminant insect-borne viruses, characterized in that: The ruminant arboviruses are AKAV, BTV, CHUV and EHDV.

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