A quadruple fluorescence quantitative RT-PCR detection kit for detecting arboviruses in ruminants, its usage method and application

Abstract

A quadruple fluorescence quantitative RT-PCR detection kit for detecting arboviruses in ruminants, its use method and application belong to the technical field of molecular biology. In order to solve the problem that there is a lack of a detection method in the prior art that is fast, sensitive, specific and can simultaneously detect BTV, EHDV, AKAV, and CHUV viruses, the present invention provides a quadruple fluorescence quantitative RT-PCR detection kit for detecting arboviruses in ruminants. The kit includes primer pairs and probe combinations, as well as a quadruple fluorescence quantitative RT-PCR detection method. The detection method has good reaction specificity, sensitivity and repeatability. The quadruple fluorescence quantitative RT-PCR detection method has a relatively high accuracy of positive rate for the detection results of AKAV, BTV, EHDV and CHUV, and can be applied to the routine detection of clinical samples and laboratory samples and the daily monitoring of ruminant populations.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a detection kit for detecting arboviruses in ruminants, a using method and an application thereof. Background Art

[0002] Bluetongue virus (BTV) belongs to the genus Orbivirus of the family Reoviridae. The virus particles have a diameter of about 80 nm and are icosahedrally symmetric. Its genome consists of 10 segments of double-stranded RNA (dsRNA). BTV is transmitted by Culicoides midges and mainly infects ruminants such as sheep, goats, and cattle, among which sheep are the most susceptible. Bluetongue disease is a legally notifiable animal disease of the World Organization for Animal Health (WOAH). The main clinical manifestations include elevated body temperature, salivation, cyanosis of the tongue, erosion or ulceration of the oral mucosa, and in severe cases, hoof shedding; infection with some serotype strains can cause abortion in pregnant female animals, production of mummified fetuses, etc., resulting in reproductive disorders. Currently, at least 29 different serotypes of BTV (BTV-1 to BTV-29) have been isolated and identified globally, as well as several BTV isolates whose serotypes have not been finally confirmed, and are tentatively identified as BTV-30 to BTV-36. Multiple different serotypes such as BTV-1, -2, -3, -4, -5, -7, -9, -12, -14, -15, -16, -20, -21 have been detected in China.

[0003] Epizootic haemorrhagic disease virus (EHDV) belongs to the genus Orbivirus of the family Reoviridae. The virus particles are icosahedrally symmetric with a diameter of about 80 nm and have a double capsid structure. Its genome consists of 10 segments of dsRNA. EHDV is transmitted among animals by blood-sucking insects such as Culicoides midges and mainly infects ruminants such as deer, cattle, and sheep, among which white-tailed deer are the most severely affected species. Epizootic haemorrhagic disease is a cross-border animal disease legally reported by WOAH. The main clinical symptoms are high fever, anorexia, weakness, swelling and cyanosis of the tongue, respiratory distress, head and neck edema, nasal mucosa congestion, nasal cavity erosion, etc. Infection with some serotype strains can cause reproductive disorders such as abortion in pregnant animals, production of dead fetuses, and fetal brain malformations. Currently, 10 different serotypes of EHDV have been discovered globally, including EHDV-1, -2, -4 to -11. Among them, multiple serotypes such as EHDV-1, -2, -5, -6, -7, -8, -10, -11 have been detected in China.

[0004] Akabane virus (AKAV) belongs to the Orthobunyavirus genus of the Bunyaviridae family and is a member of the Simbu virus serogroup. The virus particles are approximately 80 - 120 nm in diameter, spherical with an envelope, and the viral genome is single-stranded negative-sense segmented RNA, divided into three segments. AKAV is transmitted among animals through the bites of blood-sucking insects such as Culicoides and mosquitoes. Susceptible animals include domestic ruminants such as cattle, sheep, and goats, and there are also records of positive AKAV detections in animals such as wildebeest, antelopes, buffaloes, and camels. Akabane disease is a notifiable disease as defined by the WOAH. AKAV infection in adult cattle and sheep is mostly latent. The main clinical symptoms of the virus infecting fetuses are abortion, stillbirth, abnormal or deformed fetal body shapes (such as joint flexion in newborns, hydranencephaly syndrome), etc., resulting in reproductive disorders. Currently, it is believed that AKAV has only one serotype and four genetic groups (Asian group, Australian group, other group, and the fourth genetic group). Positive AKAV detection records have been found in ruminant populations in regions such as Shandong, Shanghai, Beijing, Hunan, Guangxi, Zhejiang, Fujian, Gansu, Hebei, Inner Mongolia, and Jilin in China.

[0005] Chuzan virus (CHUV) belongs to the Palyam serogroup of the Orbivirus genus of the Reoviridae family. The virus particles are approximately 50 nm in diameter, round and non-enveloped, and the viral genome consists of 10 segments of dsRNA. CHUV is transmitted among ruminants such as cattle and sheep through the bites of Culicoides, which serves as both a vector and a reservoir host. The most susceptible animals are cattle. CHUV infection in adult cattle is mostly latent with no obvious clinical symptoms; infection in pregnant cows mainly presents as reproductive disorders such as abortion, stillbirth, and the birth of deformed fetuses; infection in calves can manifest as hydranencephaly, cerebellar hypoplasia, etc. In 2013, CHUV was first isolated in Guangxi in China, 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 China's livestock and poultry breeding industry. Epidemiological investigations show that the infection rates of BTV, EHDV, AKAV, and CHUV are relatively high in the ruminant population in China, and multiple strains of BTV and EHDV with different serotypes, as well as AKAV and CHUV strains, have been isolated. First, there are a wide variety of vector insects with transmission ability 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 diffusion of virus infections. Second, in recent years, the breeding quantities of cattle, sheep, deer, etc. have increased rapidly and continuously, the susceptible animal population has been continuously expanding, and the international agricultural and livestock product trade has become increasingly frequent. All of the above factors highly alert that arbovirus diseases have posed a serious threat to China's ruminant breeding industry. Moreover, BTV, EHDV, AKAV, and CHUV have the same transmission vectors and susceptible animal populations, and their epidemic regions overlap, with the risk of mixed infection.

[0007] In the prior art, the detection methods for AKAV, BTV, CHUV, and EHDV are mostly single detections, which are difficult to meet the needs of simultaneously detecting multiple diseases; therefore, those skilled in the art are eager to develop a detection method that is rapid, sensitive, specific, and can simultaneously detect the above four viruses for daily monitoring and early differential diagnosis of diseases. Summary of the Invention

[0008] In order to solve the problem in the prior art of lacking a detection method that is rapid, sensitive, specific, and can simultaneously detect BTV, EHDV, AKAV, and CHUV viruses, the present invention provides a quadruple fluorescence quantitative RT-PCR detection kit for detecting arboviruses in ruminants, as well as a usage method and application.

[0009] One of the purposes of the present invention is to provide a quadruple fluorescence quantitative RT-PCR detection kit for detecting arboviruses in ruminants. The quadruple fluorescence quantitative RT-PCR detection kit includes primer pairs and probe combinations, and includes the following components:

[0010] The specific primer pair and probe nucleotide sequences for detecting AKAV are shown as 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 as 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 as SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9 respectively;

[0013] The nucleotide sequences of the specific primer pairs and probes for detecting EHDV are shown as 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 for AKAV, BTV, CHUV, and EHDV use the conserved sequence fragments of the AKAV genomic segment S, the BTV genomic segment S10, the CHUV genomic segment S3, and the EHDV genomic segment S9 as templates respectively.

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

[0016] In a preferred embodiment of the present invention, the addition amounts of the primer pairs are specifically as follows:

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

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

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

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

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

[0022] In a preferred embodiment of the present invention, the procedure of the quadruple fluorescence quantitative RT-PCR reaction in the described usage method is 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 56°C for 30 s; a total of 45 cycles.

[0023] In a preferred embodiment of the present invention, the method for interpreting the qualitative results is as follows: under the condition that the quality of the quadruple fluorescence quantitative RT-PCR detection is under control, if there is an amplification curve showing an S shape 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 there is an amplification curve showing an S shape or exponential growth and the CT value is greater than or equal to 39 and less than or equal to 42, the detection result is determined to be uncertain and re-extraction of nucleic acid is required for re-examination; if there is an amplification curve showing an S shape or exponential growth in the re-examination and the CT value is less than 42, the detection result is determined to be positive, otherwise it is negative.

[0024] In a preferred embodiment of the present invention, the method for interpreting the quantitative results is as follows: Gradiently dilute the plasmid standard product, convert it into the copy number according to the molecular weight of the target gene, draw a standard curve with the Ct value after amplification as the ordinate and the logarithm of the copy number as the abscissa, substitute the Ct value of the detected substance into the standard curve, and calculate the copy numbers of AKAV S, BTV S10, CHUV S3, and EHDV S9 genes for quantitative detection.

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

[0026] The beneficial effects of the present invention: The present invention provides a quadruple fluorescence quantitative RT-PCR detection kit for detecting arboviruses in ruminants. The kit includes primer pairs and probe combinations (the nucleotide sequences are shown in SEQ ID NO.1-12), as well as a quadruple fluorescence quantitative RT-PCR detection reaction system and reaction procedure; the BTV primers and probes can specifically detect at least 33 different serotypes of BTV, namely 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, namely EHDV-1, EHDV-2, EHDV-4 to EHDV-8, and EHDV-10.

[0027] The quadruple fluorescence quantitative RT-PCR detection kit provided by the present invention can only successfully amplify the target genes of AKAV, BTV, CHUV, and EHDV, and no cross-reaction occurs; the lowest detection limit of the quadruple fluorescence quantitative RT-PCR detection kit for AKAV, BTV, CHUV, and EHDV is 100 copies / reaction; the results of repeated intra-group and inter-group detections of the quadruple fluorescence quantitative RT-PCR detection kit of the present invention show that the intra-group coefficient of variation is between 0.10% and 0.92%, and the inter-group coefficient of variation is between 1.10% and 2.53%. The coefficients of variation of intra-group and inter-group repeated detections are both below 3%. In summary, the quadruple fluorescence quantitative RT-PCR detection kit and its use method provided by the present invention have good reactivity, specificity, sensitivity, and repeatability.

[0028] In the quadruple fluorescence quantitative RT-PCR detection kit provided by the present invention, the positive rates of sample detections of AKAV, BTV, CHUV, and EHDV are consistent with the published single fluorescence quantitative RT-PCR detection results; it can be seen that the quadruple fluorescence 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 the routine detection of clinical samples and laboratory samples and the daily monitoring of ruminant populations, providing a faster, more convenient, and efficient detection method for ruminant arbovirus infectious diseases, and providing a new technology for the detection of AKAV, BTV, CHUV, and EHDV. Description of the Drawings

[0029] Figure 1 It is the optimization diagram of the primer and probe concentrations of the 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;

[0030] Figure 2 It is the optimization diagram of the annealing temperature of the 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 It is the amplification curve and standard curve diagram of the AKAV single fluorescence quantitative RT-PCR detection in Example 2; A is the amplification curve diagram, and B is the standard curve diagram;

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

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

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

[0035] Figure 7 Specificity detection graph for single fluorescence quantitative RT-PCR in Example 2; A is the detection graph of AKAV, B is the detection graph of BTV, C is the detection graph of CHUV, D is the detection graph of EHDV;

[0036] Figure 8 Sensitivity detection graph for single fluorescence quantitative RT-PCR in Example 2; A is the detection graph of AKAV, B is the detection graph of BTV, C is the detection graph of CHUV, D is the detection graph of EHDV;

[0037] Figure 9 Amplification curve and standard curve graph for quadruple fluorescence quantitative RT-PCR detection in Example 3; A is the amplification curve graph, B is the standard curve graph;

[0038] Figure 10 Specificity detection graph for quadruple fluorescence quantitative RT-PCR in Example 3, A is the detection graph of the mixed nucleic acid sample of four viruses, B is the detection graph of the single virus nucleic acid sample;

[0039] Figure 11 Sensitivity detection graph for quadruple fluorescence quantitative RT-PCR in Example 3; A is the summary result graph of sensitivity detection; B is the detection graph of AKAV, C is the detection graph of BTV, D is the detection graph of CHUV, E is the detection graph of EHDV. Detailed implementation manners

[0040] Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those related can obviously make changes or appropriate alterations and combinations to 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 objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification. The test methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in this field, and those skilled in the art can obtain them through commercial channels without special instructions.

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

[0043] Virus samples: Viruses such as Akabane virus (AKAV), Bluetongue virus (BTV), Chuzan virus (CHUV), Epizootic hemorrhagic disease virus (EHDV), Peste des petits ruminants virus (PPRV), Goatpox 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. The above viruses were kindly provided by the Innovation Team of Bovine and Sheep Infectious Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0044] Plasmid samples: 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 kindly provided by the Innovation Team of Bovine and Sheep Infectious Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. A 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. The gene fragment was inserted into the pUC57 cloning vector to construct a tandem standard plasmid named pUC57-AKAV-BTV-CHUV-EHDV plasmid.

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

[0046] Instruments: Electrothermal constant temperature water bath (Shanghai Yiheng Technology Co., Ltd.), Centrifuge 5418 (Eppendorf), QuantStudio 5 Real-Time Fluorescent 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 genomic sequences of AKAV, different serotypes of BTV, CHUV, and different serotypes of EHDV were downloaded from the Genbank database respectively. After alignment and analysis, the AKAV-S segment, BTV-S10 segment, CHUV-S3 segment, and EHDV-S9 segment were selected as the 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 degenerate base C+T, R represents degenerate base A+G, K represents degenerate base G+T, W represents degenerate base A+T, M represents degenerate base A+C, and N represents 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 the genomic RNAs of AKAV, BTV-1 / 12 / 15 / 16 / 20, CHUV, EHDV-2 / 5 / 7, PPRV, GPV, BRSV, BRV, BPIV3, and IBRV according to its instructions, and they were placed in a -80°C refrigerator for standby.

[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 midiprep kit respectively. The concentrations of the above plasmids were measured using an ultra-micro ultraviolet spectrophotometer, and the copy numbers of the above plasmids were obtained respectively; then, using EASY Dilution according to a gradient of 1:10, the above plasmids were diluted to 1×10 9 -1×10 0 copies / μL and placed in a -20°C refrigerator for standby.

[0057] Example 2: Construction and Verification of a Single Fluorescent Quantitative RT-PCR Detection Reaction System

[0058] (1) Construction of a Single Fluorescent Quantitative RT-PCR Detection Reaction System

[0059] In this example, the matrix method was used to optimize the primer and probe dosages and reaction temperature in the single fluorescence quantitative RT-PCR reaction systems for AKAV, BTV, CHUV, and EHDV.

[0060] The reaction program for fluorescence quantitative RT-PCR detection was 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, with a total of 45 cycles of denaturation and extension.

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

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

[0063] On the basis of the above primer pairs and probe dosages for the single fluorescence quantitative RT-PCR reactions of AKAV, BTV, CHUV, and EHDV, and other reaction programs 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, 62 °C). The results are as Figure 2 shown, and it was determined that the optimal annealing temperature for the single fluorescence quantitative RT-PCR reactions of AKAV, BTV, CHUV, and EHDV was 56 °C.

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

[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 respectively serially diluted 10-fold. Eight different concentration gradients of 1×10 8 copies / μL - 1×10 1 copies / μL were respectively used as templates for single fluorescence quantitative RT-PCR amplification reactions. Standard curves were respectively prepared. Among them, the single fluorescence quantitative standard curve of AKAV was y = -3.197log(X) + 38.345, R 2 = 0.998, Eff% = 105.496 (as Figure 3 shown); the single fluorescence quantitative standard curve of BTV was y = -3.314log(X) + 38.800, R 2 = 0.998, Eff% = 100.324 (as Figure 4 shown); the single fluorescence quantitative standard curve of CHUV was y = -3.293log(X) + 39.507, R 2 = 1.000, Eff% = 101.213 (as Figure 5 shown); the single fluorescence quantitative standard curve of EHDV was y = -3.297log(X) + 39.446, R 2 = 0.999, Eff% = 101.059 (as Figure 6 shown).

[0066] It can be seen that the single fluorescence quantitative RT-PCR reaction systems of AKAV, BTV, CHUV, and EHDV provided by the present invention have good linear relationships within the range of eight different concentration gradients of 1×10 8 copies / μL - 1×10 1 copies / μL.

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

[0068] Using the genomes of AKAV, BTV-1 / 12 / 15 / 16 / 20, CHUV, and EHDV-2 / 5 / 7 as positive nucleic acid samples, and using the genomic RNAs of PPRV, GPV, BRSV, BRV, BPIV3, and IBRV obtained in Example 1 as control samples, the specificity verification of single fluorescence quantitative RT-PCR detection was respectively carried out on the above samples using the primers and probes of four pathogens, AKAV, BTV, CHUV, and EHDV.

[0069] The results are as Figure 7As shown, only four target genes, namely AKAV, BTV, CHUV, and EHDV, were successfully amplified without cross-reaction, demonstrating 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 serially diluted 10-fold. Ten different concentration gradients of plasmids from 1×10 9 copies / μL to 1×10 0 copies / μL were used as templates for single fluorescence quantitative RT-PCR amplification reactions. At the same time, plasmids at 1×10 1 copies / μL and 1×10 0 copies / μL were used as templates for 28 repeated detections. With a CT value ≤ 39 defined as positive, the positive detection rate of the 28 repeated detections was calculated. If the positive detection rate ≥ 75%, then this concentration was determined to be the detectable concentration.

[0072] The sensitivity test results are as Figure 8 shown in Table 2. The minimum detection limit of the single fluorescence quantitative RT-PCR detection methods 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, four dilution concentrations of plasmids from 1×10 6 copies / μL to 1×10 3 copies / μL of the pCI-AKAV-S plasmid, pCI-BTV-S10 plasmid, pCI-CHUV-S3 plasmid, and pCI-EHDV-S9 plasmid obtained in (1) were selected for single fluorescence quantitative RT-PCR repeatability detection, with intra-group and inter-group repeated detections. For the intra-group test, three replicate experiments were performed for each dilution concentration of the plasmid in a group; the above experiments were repeated three times as the inter-group test.

[0077] The results of repeatability verification are shown in Table 3. The within-group coefficient of variation is between 0.17% and 1.69%, and the between-group coefficient of variation is between 0.36% and 2.53%. The coefficients of variation of repeated detections within and between groups are 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 program in the quadruple fluorescence quantitative RT-PCR detection kit

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

[0083] The pUC57-AKAV-BTV-CHUV-EHDV plasmid obtained in Example 1 was serially diluted 10-fold. The quadruple fluorescence quantitative RT-PCR detection reaction system is shown in Table 4. Among them, the concentrations of primers and probes are both 10 μM. The program of the quadruple fluorescence quantitative RT-PCR reaction 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 the standard curve of the quadruple fluorescence quantitative RT-PCR detection kit

[0088] Eight plasmid templates with different concentration gradients from 1×10 9 copies / μL to 1×10 2 copies / μL obtained in (1) were selected as templates for quadruple fluorescence quantitative RT-PCR reaction. The results are as Figure 9 shown.

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

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

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

[0092] The multiplex fluorescence quantitative standard curve of EHDV 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] Using the genomes of AKAV, BTV-1 / 12 / 15 / 16 / 20, CHUV, and EHDV-2 / 5 / 7 as positive nucleic acid samples, and using the genomes of PPRV, GPV, BRSV, BRV, BPIV3, and IBRV as controls, the specificity of the quadruple fluorescence quantitative RT-PCR detection kit was verified using the primers and probes of the four pathogens respectively.

[0096] The results are as Figure 10 shown. Only the 4 target genes of AKAV, BTV, CHUV, and EHDV were successfully amplified without cross-reaction, proving 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 copies / μL of 10 different concentration gradients (1) of the pUC57-AKAV-BTV-CHUV-EHDV plasmid obtained were used as templates for the amplification reaction of quadruple fluorescence quantitative RT-PCR. At the same time, 1×10 2 copies / μL plasmid and 1×10 1 copies / μL plasmid were used as templates for 28 repeated detections. With CT value ≤ 39 as positive, the positive detection rate of the 28 repeated detections was calculated. If the positive detection rate ≥ 75%, then the concentration was determined as the detectable concentration.

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

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

[0101] Table 5

[0102]

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

[0104] In this example, the pUC57-AKAV-BTV-CHUV-EHDV plasmid obtained at a total of 4 dilution concentrations of 1×10 6 copies / μL - 1×10 3 copies / μL was used for multiple fluorescence quantitative RT-PCR repeatability detection, and intra-group and inter-group repeat detections were carried out; 3 replicate tests were performed on the plasmid at each dilution concentration in one group as the intra-group test; the above experiment was repeated 3 times as the inter-group test.

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

[0106] Table 6

[0107]

[0108]

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

[0110] In this example, to verify the feasibility of the quadruple fluorescence quantitative RT-PCR detection kit, the quadruple fluorescence quantitative RT-PCR detection kit was used to detect 138 samples (including clinical cattle and sheep blood samples and laboratory samples). At the same time, the above samples were detected respectively by using the publicly available single fluorescence quantitative RT-PCR detection methods for AKAV, BTV, CHUV and EHDV, and the detection results were compared; among them, the publicly available single fluorescence quantitative RT-PCR detection method for BTV referred to the Bluetongue diagnosis technology of GB / T 18636-2017, the publicly available single fluorescence quantitative RT-PCR detection method for AKAV referred to the Real-time fluorescence RT-PCR detection method for Akabane virus of SN_T 3991-2014, and the publicly available single fluorescence quantitative RT-PCR detection methods for EHDV and CHUV referred to the 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 methods for epizootic hemorrhagic disease virus). Transbound Emerg Dis. 2017 Aug;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] As shown in Table 7, when the quadruple fluorescence quantitative RT-PCR detection kit provided by the present invention was used for sample detection, among them, 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 for Akabane virus of 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 publicly published research paper of CHUV; the positive rate of EHDV was 9.42% (13 / 138), which was consistent with the detection result of the detection method in the publicly published research paper of EHDV.

[0112] This indicates that the detection results of the quadruple fluorescence quantitative RT-PCR detection kit provided by the present invention have a relatively high coincidence rate with the detection results of 4 publicly disclosed single fluorescence quantitative RT-PCR detection methods for AKAV, BTV, CHUV, and EHDV. Therefore, the quadruple fluorescence 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 the routine detection of clinical samples and laboratory samples and the daily monitoring of ruminant populations, providing a faster, more convenient, and more efficient detection method for ruminant arboviral infectious diseases.

[0113] Table 7

[0114]

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

Claims

1. A quadruple fluorescence quantitative RT-PCR detection kit for detecting arboviruses in ruminants, characterized in that, The ruminant arboviruses are AKAV, BTV, CHUV, and EHDV. The quadruple fluorescence quantitative RT-PCR detection kit includes primer pairs and probe combinations, and comprises the following components: The nucleotide sequences of the specific primer pair and probe for detecting AKAV are shown as SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively; The nucleotide sequences of the specific primer pair and probe for detecting BTV are shown as SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 respectively; The nucleotide sequences of the specific primer pair and probe for detecting CHUV are shown as SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9 respectively; The nucleotide sequences of the specific primer pair and probe for detecting EHDV are shown as SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12 respectively.

2. The quadruple fluorescence quantitative RT-PCR detection kit according to claim 1, wherein, The specific primer pairs and probes for AKAV, BTV, CHUV, and EHDV use the conserved sequence fragments of the AKAV genome segment S, the BTV genome segment S10, the CHUV genome segment S3, and the EHDV genome segment S9 as templates respectively.

3. Method for non-diagnostic use of the quadruple fluorescence quantitative RT-PCR detection kit according to any one of claims 1-2, characterized in that, The usage method includes the following steps: Using the total nucleic acid extracted from the sample to be detected as a template, performing quadruple fluorescence quantitative RT-PCR reaction with the four specific primer pairs and probe sequences in the quadruple fluorescence quantitative RT-PCR detection kit, and the quadruple fluorescence quantitative RT-PCR reaction system, and finally interpreting the qualitative and quantitative results.

4. The usage method according to claim 3, characterized in that, The program of the quadruple fluorescence quantitative RT-PCR reaction in the usage method 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.

5. The usage method according to claim 3, wherein, The method for interpreting the qualitative results is as follows: Under the condition that the quality of the quadruple fluorescence quantitative RT-PCR detection is under control, if there is an amplification curve showing an S shape 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 there is an amplification curve showing an S shape or exponential growth and the CT value is greater than or equal to 39 and less than or equal to 42, the detection result is determined to be uncertain, and nucleic acid needs to be re-extracted for re-examination; If there is an amplification curve showing an S shape or exponential growth and the CT value is less than 42 in the re-examination, the detection result is determined to be positive, otherwise it is negative.

6. The usage method according to claim 3, characterized in that, The method for interpreting the quantitative results is as follows: Gradiently dilute the plasmid standard product, convert it into the copy number according to the molecular weight of the target gene, draw a standard curve with the Ct value after amplification as the ordinate and the logarithm of the copy number as the abscissa, substitute the Ct value of the sample to be detected into the standard curve, and calculate the copy numbers of the AKAV S, BTV S10, CHUV S3, and EHDV S9 genes for quantitative detection.

7. Use of the quadruple fluorescence quantitative RT-PCR detection kit according to any one of claims 1-2 in the detection of arboviruses in ruminants for non-diagnostic purposes, characterized in that, The ruminant arboviruses are AKAV, BTV, CHUV, and EHDV.

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