A dual fluorescence RT-LAMP detection kit for chicken astrovirus and avian nephritis virus

Abstract

The invention discloses a dual fluorescence RT-LAMP detection kit for chicken astrovirus and avian nephritis virus, and relates to the field of biological detection technology. The kit includes a primer and a probe combination, specifically including primers CAstV-F3, primers CAstV-B3, probes CAstV-FIP, primers CAstV-BIP, primers CAstV-Floop, primers CAstV-Bloop, probes CAstV-FD, primers ANV-F3, primers ANV-B3, probes ANV-FIP, primers ANV-BIP, primers ANV-Floop, primers ANV-Bloop and probes ANV-FD, whose nucleotide sequences are shown in SEQ ID NO.1-14, respectively. The kit has fast, accurate detection, good specificity, high sensitivity and good repeatability, and can provide technical support for epidemiological surveys of CAstV and ANV.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, in particular to a double fluorescence RT-LAMP detection kit for chicken astrovirus and avian nephritis virus. Background Art

[0002] Chicken astrovirus (CAstV) and avian nephritis virus (ANV) are two common astroviruses (Astrovirus, AstV) from chickens. So far, there are no effective drugs and vaccines for the treatment of CAstV and ANV. The only way to reduce the impact is to rely on measures such as regular disinfection and chicken purification. Therefore, an effective clinical detection technology is urgently needed to provide technical support for virus monitoring. There are currently no reports of large-scale outbreaks of the two viruses, but in clinical practice, CAstV and ANV often cause mixed infections. Infection in laying hens will cause a decrease in egg production. Both viruses can be transmitted vertically through chicken embryos. Infection in broilers can cause symptoms such as developmental disorders, causing economic losses to the poultry industry. Therefore, in order to be able to perform early diagnosis and disease monitoring of the two viruses, a practical method is needed for clinical detection.

[0003] At present, the detection technologies for CAstV and ANV mainly include virus isolation, RT-PCR, fluorescence quantitative PCR, nested RT-PCR, indirect ELISA, etc. The above detection methods all have disadvantages to varying degrees: virus isolation is accurate and reliable, but time-consuming and lacks ideal cell lines; PCR operation is simple but time-consuming, and the product is easy to remain and cause false positives; fluorescence quantitative PCR has high sensitivity, but the instrument requires precision and its grassroots application is limited; ELISA method is easy to operate, but false positives are prone to occur during the detection process and can only detect one virus at a time. With the advancement of technology, new detection technologies such as loop-mediated isothermal amplification (LAMP) have been introduced into poultry disease detection to achieve efficient and convenient diagnosis. This method has the advantages of simple operation, short reaction time, constant temperature water bath reaction, and no need for expensive equipment.

[0004] The present invention intends to develop a dual fluorescence RT-LAMP detection kit for chicken astrovirus and avian nephritis virus, thereby providing technical support for the epidemiological investigation of chicken astrovirus and avian nephritis virus. Summary of the invention

[0005] The purpose of the present invention is to provide a dual fluorescence RT-LAMP detection kit for chicken astrovirus and avian nephritis virus to solve the problems existing in the above-mentioned prior art. The kit has the characteristics of rapidity, accuracy, good specificity, high sensitivity, good repeatability, etc., is suitable for clinical diagnosis, and can provide technical support for epidemiological investigations of CAstV and ANV.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a primer and probe combination for double fluorescence RT-LAMP detection of chicken astrovirus and avian nephritis virus, comprising a primer CAstV-F3, a primer CAstV-B3, a probe CAstV-FIP, a primer CAstV-BIP, a primer CAstV-Floop, a primer CAstV-Bloop, a probe CAstV-FD, a primer ANV-F3, a primer ANV-B3, a probe ANV-FIP, a primer ANV-BIP, a primer ANV-Floop, a primer ANV-Bloop and a probe ANV-FD, the nucleotide sequences of which are respectively shown as SEQ ID NO.1-14.

[0008] Furthermore, the 3'-ends of the probe CAstV-FD ​​and the probe ANV-FD are labeled with different fluorescent groups;

[0009] The 5' ends of the probe CAstV-FIP and the probe ANV-FIP are labeled with different quenching groups.

[0010] Furthermore, the 3' end of the probe CAstV-FD ​​is labeled with a FAM group;

[0011] The 3' end of the probe ANV-FD is labeled with a CY3 group;

[0012] The 5' end of the probe CAstV-FIP is labeled with a BHQ1 group;

[0013] The 5' end of the probe ANV-FIP is labeled with a BHQ2 group.

[0014] The present invention also provides a primer and probe combination for chicken astrovirus fluorescent RT-LAMP detection, comprising primer CAstV-F3, primer CAstV-B3, probe CAstV-FIP, primer CAstV-BIP, primer CAstV-Floop, primer CAstV-Bloop and probe CAstV-FD, whose nucleotide sequences are respectively shown as SEQ ID NO.1-7.

[0015] Furthermore, the 3' end of the probe CAstV-FD ​​is labeled with a fluorescent group;

[0016] The 5' end of the probe CAstV-FIP is labeled with a quenching group.

[0017] The present invention also provides a primer and probe combination for avian nephritis virus fluorescent RT-LAMP detection, including primer ANV-F3, primer ANV-B3, probe ANV-FIP, primer ANV-BIP, primer ANV-Floop, primer ANV-Bloop and probe ANV-FD, whose nucleotide sequences are respectively shown as SEQ ID NO.8-14.

[0018] Furthermore, the 3' end of the probe ANV-FD is labeled with a fluorescent group;

[0019] The 5' end of the probe ANV-FIP is labeled with a quenching group.

[0020] The present invention also provides the use of the primer and probe combination for dual fluorescence RT-LAMP detection of chicken astrovirus and avian nephritis virus in preparing a dual fluorescence RT-LAMP detection kit for chicken astrovirus and avian nephritis virus.

[0021] The invention also provides a dual fluorescence RT-LAMP detection kit for chicken astrovirus and avian nephritis virus, comprising a primer and probe combination for dual fluorescence RT-LAMP detection of chicken astrovirus and avian nephritis virus.

[0022] Further, the reaction system of the dual fluorescence RT-LAMP detection kit is: 2×Master Mix 12.5 μL, Bst DNA polymerase 0.1 μL, primers CAstV-F3, CAstV-B3, ANV-F3 and ANV-B3 0.5 μL each, primers CAstV-F3, CAstV-B3, ANV-F3 and ANV-B3 0.5 μL each, primers CAstV-Floop, CAstV-Bloop, ANV-Floop and ANV-Bloop 0.5 μL each, primers CAstV-BIP and ANV-BIP 0.5 μL each, FIP-FD 0.5 μL, DNA / cDNA template 2 μL, RNase-free Water 2.4 μL;

[0023] The FIP-FD includes 0.48 μmol / L CAstV-FIP-FD and 0.32 μmol / L A StV-FIP-FD;

[0024] The CAstV-FIP-FD is a composite probe formed by annealing the probe CAstV-FIP and the probe CAstV-FD;

[0025] The ANV-FIP-FD is a composite probe formed by annealing the probe ANV-FIP and the probe ANV-FD.

[0026] The present invention discloses the following technical effects:

[0027] According to the conserved sequences of CAstV ORF1b gene and ANV ORF1b gene, the present invention respectively designs two sets of specific primers and probes labeled with fluorescent groups. The 3' end of CAstV probe is labeled with FAM fluorescent group and the 5' end is labeled with quenching group BHQ1; the 3' end of ANV probe is labeled with CY3 fluorescent group and the 5' end is labeled with quenching group BHQ2. The most suitable primers and probes are screened out through experiments and the reaction conditions are optimized. The established dual fluorescence RT-LAMP was used to conduct specificity, sensitivity, repeatability and interference tests, and a total of 300 clinical samples collected from different regions of Nanning City, Guangxi Zhuang Autonomous Region were tested. The results were compared with RT-qPCR and RT-PCR to verify the effectiveness of the method. The test results show that the dual fluorescence RT-LAMP method established by the present invention can complete the reaction in 60 minutes, and the optimal reaction temperature is 65°C; the designed primers can only detect CAstV and ANV, and have no cross-reaction to other pathogens; the minimum detection limit of CAstV is 1.4×10 2 copies / μL, and the minimum detection limit of ANV was 1.5×10 2 The detection results of CAstV and ANV were 97.97% and 98.85% respectively compared with those of RT-qPCR. In summary, the dual fluorescence RT-LAMP method established in the present invention has the characteristics of rapidity, accuracy, good specificity, high sensitivity, good repeatability, etc. It is suitable for clinical diagnosis and can provide technical support for epidemiological investigations of CAstV and ANV. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The magnesium phosphate curve of the byproducts of the CAstV probe gradient screening experiment was monitored by a real-time turbidometer;

[0030] Figure 2 The magnesium phosphate curve of the byproducts of the ANV probe gradient screening experiment was monitored by a real-time turbidometer;

[0031] Figure 3 It is the fluorescence image of CAstV in the 520nm channel; wherein, the upper row 1 to 6 represent 0% FD, 20% FD, 40% FD, 60% FD, 80% FD and 100% FD respectively; the lower row 1 to 6 are negative controls;

[0032] Figure 4 It is the fluorescence image of ANV under 570nm channel; wherein, the upper row 1 to 6 represent 0% FD, 20% FD, 40% FD, 60% FD, 80% FD and 100% FD respectively; the lower row 1 to 6 are negative controls;

[0033] Figure 5 This is the result of the dual fluorescence RT-LAMP optimal reaction temperature screening experiment;

[0034] Figure 6 This is a graph showing the results of a double fluorescence RT-LAMP specificity test; among them, 1: negative control; 2: CAstV; 3: ANV; 4: CAstV+ANV; 5: IBV; 6: AGV2; 7: GyV3; 8: ChPV; 9: FAdV-4; 10: CIAV; 11: AIV-H5; 12: AIV-H7; 13: HEV; 14: NDV-6; 15: NDV-7; 16: MS; 17: MG; 18: ALV; 19: ARV;

[0035] Figure 7 The results of the dual fluorescence RT-LAMP detection of CAstV single template sensitivity experiment; A is the curve of the magnesium phosphate byproduct of CAstV monitored by the real-time turbidity meter; B is the image of the CAstV single template reaction product under the multi-color imaging analysis system; in B, 2~10:10 9 ~10 1 copies / μL; 1: negative control (RNase-free Water);

[0036] Figure 8 The results of the double fluorescence RT-LAMP detection of ANV single template sensitivity experiment; A is the ANV byproduct magnesium phosphate curve monitored by the real-time turbidity meter; B is the image of the ANV single template reaction product under the multi-color imaging analysis system; in B, 2~10:10 9 ~10 1 copies / μL; 1: negative control (RNase-free Water);

[0037] Fig. 9 RT-PCR sensitivity electrophoresis diagram; A is CAstV RT-PCR sensitivity electrophoresis diagram; B is ANV RT-PCR sensitivity electrophoresis diagram; in A and B, M: DNA molecular mass standard; 2~10:109 ~10 1 copies / μL plasmid standard; 1: negative control (RNase-free Water);

[0038] Fig.10 The results of the dual fluorescence RT-LAMP sensitivity experiment for detecting CAstV and ANV mixed templates; A is the curve of byproduct magnesium phosphate monitored by the real-time turbidity meter; B is the image of the reaction product of CAstV and ANV under the multi-color imaging analysis system; in B, a is the fluorescence signal of the mixed template reaction product of CAstV and ANV at 520nm channel; b is the fluorescence signal of the mixed template reaction product of CAstV and ANV at 570nm; c is the fluorescence signal of the mixed template reaction product of CAstV and ANV at 520nm channel and 570nm channel; in a, b and c, 1: negative control (RNase-free Water); 2-10: 10 9 ~10 1 copies / μL;

[0039] Fig.11 The results of the double fluorescence RT-LAMP interference test are shown in Figure 2. Among them, 2-10: ANV is 10 9 copies / μL, CAstV were 10 9 ~10 1 copies / μL; 12-20: CAstV is 10 9 copies / μL, ANV was 10 9 ~10 1 copies / μL; 1 and 11: negative control (RNase-free Water). DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Example 1

[0046] 1 Materials and methods

[0047] 1.1 strains

[0048] The control samples used in the experiment were all DNA or cDNA, and the specific information is shown in Table 1. All nucleic acid samples have been verified by PCR and sequenced to be correct.

[0049] Table 1 Strain information

[0050]

[0051]

[0052] 1.2 Test materials and instruments

[0053] Loopamp LA-320C real-time turbidity meter was purchased from Rongyan Biotechnology Co., Ltd.; multicolor imaging analysis system, Gel Doc TM XR+ gel imager was purchased from Bio-Rad, USA; Nano Drop 2000 UV spectrophotometer was purchased from Thermo Fisher Scientific, USA; DNA / RNA virus extraction kit was purchased from Beijing Quanshijin Company; pMD-18T vector, reverse transcription kit, ampicillin and premix Taq PCR reagent were purchased from Bao Biotechnology (Dalian) Co., Ltd.; DH5α competent cells were purchased from Biotech (Shanghai); plasmid extraction kit was purchased from Omega Biotechnology, USA.

[0054] 1.3 Design and synthesize primers and probes

[0055] MEGA 11 software was used to compare 57 CAstV ORF1b gene sequences and 78 ANV ORF1b gene sequences downloaded from GenBank. Two sets of primers and probes for specific amplification of CAstV and ANV were designed using the LAMP primer online design website PrimerExplorer V5 https: / / primerexplorer.jp / e / index.html. The primer set included outer primers F3 and B3, inner primers FIP (FIP = F1c + F2) and BIP (BIP = B1c + B2). Based on F1c in the inner primer FIP, a probe FD complementary to F1c was synthesized. The probe FD was modified with a fluorescent group at the 3′ end, and FIP was modified with a quenching group at the 5′ end. Before the reaction, FIP and FD were annealed at 95°C to form a composite probe (FIP-FD). Since FD was complementary to the F1c fragment in the FIP inner primer, the fluorescent group was close to the quenching group, resulting in the disappearance of fluorescence. At the beginning of the reaction, FIP and FD separated, resulting in the release of the fluorescent group. The 3′ end of CAstV-FD ​​is labeled with a FAM group, which emits green fluorescence at a wavelength of 520 nm, and the 5′ end of CAstV-FIP is labeled with a quencher group BHQ1. The 3′ end of ANV-FD is labeled with a CY3 fluorescent group, which emits blue fluorescence at a wavelength of 570 nm, and the 5′ end of ANV-FIP is labeled with a quencher group BHQ2. The primers were synthesized by Sangon (Shanghai) and purified to grade HPL C (Table 2).

[0056] Table 2 Fluorescent RT-LAMP primer and probe sequence list

[0057]

[0058]

[0059] 1.4 Nucleic acid extraction and preparation of positive standards

[0060] According to the instructions of the viral DNA / RNA extraction kit, nucleic acid was extracted from CAstV and ANV, and reverse transcribed into cDNA and stored in a -80℃ refrigerator for later use. The extracted cDNA was used to amplify the conservative sequences of CAstV and A NV ORF1b genes respectively using the PCR method, and the purified PCR products were collected and connected with the pMD-18T vector and transformed into competent cells DH5α. The results were verified by double enzyme digestion and sequencing by Bioengineering (Shanghai). The recombinant plasmids (named: pMD-18T-CAstV and pMD-18T-ANV) were obtained using a small amount of plasmid extraction kit. The concentration of the extracted plasmid was determined using a NanoDrop 2000 UV spectrophotometer, and the copy number was calculated according to the formula: copy number (copies / μL) = plasmid concentration (g / μL) × 10 -9 ×6.02×10 23 / 660×3300 to calculate the copy number. Finally, the prepared positive standard was placed in a -80°C refrigerator for later use.

[0061] 1.5 Establishment of dual fluorescence RT-LAMP method and optimization of reaction system

[0062] 1.5.1 Optimal probe concentration for dual fluorescence RT-LAMP

[0063] The constructed pMD-18T-ANV and pMD-18T-CAstV positive plasmids were used as standards to establish a dual fluorescence RT-LAMP reaction system. The initial reaction system was 25 μL: 12.5 μL 2×Master Mix, 0.1 μL Bst DNA polymerase, 0.5 μL each of outer primers CAstV-F3, CAstV-B3, ANV-F3 and ANV-B3 (working concentration 0.1 μmol / L), 0.5 μL each of loop primers CAstV-F3, CAstV-B3, ANV-F3 and ANV-B3 (working concentration 0.1 μmol / L), 0.5 μL each of CAstV-Flo op, CAstV-Bloop, ANV-Floop and ANV-Bloop (working concentration 0.2 μmol / L), 0.5 μL each of inner primers CAstV-BIP and ANV-BIP (working concentration 0.8 μmol / L), 0.5 μL of FIP-FD, 2 μL of DNA / cDNA template, and 2.4 μL of RNase-free Water.

[0064] According to the determined reaction system, different concentrations of FIP-FD composite probes were set up, namely (20% FD: 0.16μmol / L; 40% FD: 0.32μmol / L; 60% FD: 0.48μmol / L; 80% FD: 0.64μmol / L; 100% FD: 0.8μmol / L) to explore the optimal probe concentration in the single CAstV RT-LAMP. The ANV operation method is consistent with that of CAstV to explore the optimal probe concentration.

[0065] 1.5.2 Exploration of the optimal temperature for dual fluorescence RT-LAMP reaction

[0066] The optimal reaction temperature of dual fluorescence RT-LAMP was explored using the determined concentrations of CAstV and ANV FIP-FD. The reaction temperature of LAMP is generally between 60 and 70°C, based on which the positive template was added to the reaction system. Initial reaction procedure: 58°C for 60 minutes, inactivation at 80°C for 5 minutes. The reaction temperature of dual fluorescence RT-LAMP was increased by 1°C each time from 58°C for testing. After the reaction, the reaction tube was placed in a multicolor imaging analysis system for determination, and the optimal reaction temperature was determined based on the intensity of the fluorescence reaction.

[0067] 1.6 Dual fluorescence RT-LAMP specificity assay

[0068] Using the optimized dual fluorescence RT-LAMP reaction method 1.5, specificity tests were carried out on common poultry viruses (IBV, AGV2, GyV3, ChPV, FAdV-4, CIAV, AIV-H5, AIV-H7, HEV, NDV-6, NDV-7, MS, MG, ALV and ARV), and negative controls were set up to explore the specificity of the dual fluorescence RT-LAMP method.

[0069] 1.7 Dual fluorescence RT-LAMP sensitivity test

[0070] 1.7.1 Single pathogen template sensitivity in the dual system

[0071] According to the calculated copy number, the samples were serially diluted to 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1Based on the optimized reaction system, 2 μL of CAstV single template of different concentrations were taken and placed in separate reaction tubes, and negative controls were set. After vortex mixing, the reaction tubes were placed in a real-time turbidity meter for reaction. The above operation was repeated for ANV single template, and the test results were compared with RT-PCR to explore the sensitivity of single templates under the dual system.

[0072] 1.7.2 Dual pathogen template sensitivity in dual system

[0073] The gradient diluted ANV and CAstV plasmids were mixed in equal proportions, and then 2 μL of mixed plasmids of different concentrations were taken and placed in reaction tubes according to the preset reaction system, and negative controls were set up. After mixing, these reaction tubes were placed in a real-time turbidity meter for reaction to evaluate the sensitivity of simultaneous detection of the two pathogens.

[0074] 1.8 Dual fluorescence RT-LAMP interference test

[0075] The concentration of ANV positive template was kept constant, and the concentration of CAstV positive template was diluted in a gradient manner to perform a double fluorescence RT-L AMP reaction to explore whether the high concentration template would inhibit the amplification of the low concentration template when the concentrations of the two pathogens were different. 1 μL of each template was added, and the system was supplemented with RNase-free water to perform an interference test.

[0076] 1.9 Dual fluorescence RT-LAMP repeatability test

[0077] Select three dilutions of pMD-18T-CAstV and pMD-18T-ANV recombinant plasmid standards (10 9 , 10 5 , 10 2 The optimized conditions were used to perform the double fluorescence RT-LAMP for three replicates at the same time. At three different time points, pMD-18T-CAstV and pMD-18T-ANV recombinant plasmid standards with different dilutions were repeated three times per gradient, and double fluorescence RT-LAMP was repeated between batches. The results were statistically analyzed to determine the repeatability of the method.

[0078] 1.10 Dual fluorescence RT-LAMP clinical sample detection

[0079] A total of 300 oral and anal swab samples were collected from chickens in different areas of Nanning, Guangxi Zhuang Autonomous Region. Nucleic acids were extracted using an automated nucleic acid extractor and reverse transcribed into cDNA. The CAstV and ANV dual fluorescence RT-LAMP method, which had been optimized at 1.5, was used for detection and compared with the results of RT-PCR and RT-qPCR. The positive samples detected were sent for sequencing to verify the reliability of the method.

[0080] 2 Results

[0081] 2.1 Results of optimization of dual fluorescence RT-LAMP reaction conditions

[0082] 2.1.1 Optimal probe concentration for dual fluorescence RT-LAMP

[0083] After vortex mixing, the reactants of 25 μL system were placed in a real-time turbidity meter for reaction and negative control was set. After the reaction, the reactants were placed in a multicolor imaging analysis system for observation. It was found that ANV could react even when the probe concentration reached 100%, and the reaction time was within 60 minutes. The same result was also found for CAstV. Although the reaction could be carried out using 100% probe concentration, it was found that the inhibition reaction increased greatly with the increase of probe concentration ( Figure 1 and Figure 2 ). By comparing the start time of the real-time turbidity meter reaction and the images from the multicolor imaging analysis system, it was found that the ANV positive reaction tube showed green fluorescence. As the probe concentration increased, the fluorescence gradually increased. When the concentration reached 60%, it could achieve the purpose of distinguishing positive and negative; the CAstV positive reaction tube showed blue fluorescence ( Figure 3 and Figure 4 ). As the probe concentration increases, the fluorescence gradually increases. When the concentration reaches 40%, it can achieve the purpose of distinguishing positive and negative. Therefore, the probe concentrations of 60% CAstV and 40% ANV were selected as the optimal probe concentrations for subsequent experiments.

[0084] 2.1.2 Optimal reaction temperature of dual fluorescence RT-LAMP

[0085] The determined optimal concentrations of ANV and CAstV primers were mixed at a ratio of 1:1 and placed in a reaction tube for dual fluorescence RT-LAMP. The real-time turbidity meter and multicolor imaging analysis system showed that under the same conditions, the reaction start time was the shortest at 65°C, and the positive and negative contrast was obvious ( Figure 5 ). Therefore, 65°C was selected as the optimal reaction temperature for subsequent experiments.

[0086] 2.2 Results of dual fluorescence RT-LAMP specificity test

[0087] The nucleic acids of the pathogens listed in Table 1 were used for the double fluorescence RT-LAMP specificity test. 2 μL of template was added to each reaction tube and placed in the Loopamp LA-320C real-time turbidity meter for reaction. The results showed that the established double fluorescence RT-LAMP method only amplified CAstV, ANV, and ANV and CAstV mixed templates, while other common poultry viruses and negative controls had no amplification reaction ( Figure 6 ). In the multicolor imaging analysis system, the CAstV positive reaction tube exhibited green fluorescence in the 520nm channel; the ANV positive reaction tube exhibited dark blue fluorescence in the 570nm channel; the CAstV and ANV mixed reaction tube exhibited corresponding colors in both channels, and was cyan in the case of dual-channel superposition. The test results showed that the method of the present invention had good specificity.

[0088] 2.3 Results of dual fluorescence RT-LAMP sensitivity test

[0089] 2.3.1 Single pathogen template sensitivity in the dual system

[0090] The dual fluorescence RT-LAMP method established by the present invention was used to detect the sensitivity of ANV and CAstV single templates respectively. The Loopamp LA-320C real-time turbidity meter and the multicolor imaging analysis system both showed that when detecting ANV or CAstV positive templates alone, the minimum detection limit was 10 2 The lowest detection limit of single-plex RT-PCR for ANV and CAstV was only 10 3 copies / μL( Figure 7-Figure 9 ).

[0091] 2.3.2 Dual pathogen template sensitivity in the dual system

[0092] The sensitivity of ANV and CAstV mixed templates was tested by the established dual fluorescence RT-LAMP. The LoopampLA-320C real-time turbidity meter and multicolor imaging analysis system showed that the minimum detection limit of the dual system was 10 2 copies / μL, no interference will occur ( Fig.10 ). The results showed that the test had good sensitivity.

[0093] 2.4 Results of dual fluorescence RT-LAMP interference test

[0094] The concentration of ANV positive plasmid used was 10 9 The copies / μL remains unchanged, and the CAstV positive plasmid concentration is 10 9 ~10 1 copies / μL, the minimum detection limit of CAstV is 102 The concentration of CAstV positive plasmid was 10 copies / μL. 9 The copies / μL remains unchanged, and the ANV positive plasmid concentration is 10 9 ~10 1 copies / μL, the minimum detection limit of ANV is 10 2 The experiment proved that high-concentration templates would not interfere with the reaction of low-concentration templates, indicating that the established dual-fluorescence RT-LAMP method has good amplification results for templates of different concentrations ( Fig.11 ).

[0095] 2.5 Results of the Dual Fluorescence RT-LAMP Repeatability Test

[0096] Select three dilutions of recombinant plasmid standard (10 9 , 10 5 , 10 2 Each gradient was repeated 3 times, and 3 intra-batch and inter-batch replicates were performed.

[0097] The obtained Time value results were statistically analyzed, and the results showed that the standard deviation of the CAstV intra-batch repeat test was between 0.125 and 0.245, and the coefficient of variation was 0.67% to 0.75%; the standard deviation of the inter-batch repeat test was between 0.027 and 0.125, and the coefficient of variation was 0.15% to 0.37% (Table 3). The standard deviation of the ANV intra-batch repeat test was between 0.170 and 0.450, and the coefficient of variation was 0.47% to 1.71%; the standard deviation of the inter-batch repeat test was between 0.027 and 0.063, and the coefficient of variation was 0.15% to 0.17% (Table 4). The results showed that the dual fluorescence RT-LAMP rapid identification detection method established in this study has good repeatability.

[0098] Table 3 CAstV repeatability test results

[0099]

[0100] Table 4 ANV repeatability test results

[0101]

[0102] 2.6 Double fluorescence RT-LAMP clinical sample test results

[0103] A total of 300 oro-anal swab samples of chickens were collected from different areas of Nanning City, Guangxi Zhuang Autonomous Region, and nucleic acids were extracted using an automated nucleic acid extractor and reverse transcribed into cDNA. The established CAstV and ANV dual fluorescence RT-LAMP method was used for detection, and the results were compared with RT-PCR and RT-qPCR results, and the results are shown in Table 5. The dual fluorescence RT-LAMP method established by the present invention detected 97 (32.33%) CAst V positive samples, 86 (28.66%) ANV positive samples and 46 (15.33%) mixed infection samples, RT-PCR detected 95 (31.66%) CAstV positive samples, 80 (26.66%) ANV positive samples and 43 (14.33%) mixed infection samples; RT-qPCR detected 99 (33%) CAstV positive samples, 87 (29%) ANV positive samples and 46 (15.33%) mixed infection samples. Compared with RT-qPCR, the detection results of CAstV and ANV by RT-LAMP were consistent with 97.97% and 98.85% respectively; compared with RT-PCR and RT-qPCR, the detection results of CAstV and ANV were consistent with only 92.92% and 91.95%. The results showed that the dual fluorescence RT-LAMP method had a higher detection rate than ordinary RT-PCR and had a high consistency with the RT-qPCR results.

[0104] Table 5 Results of dual fluorescence RT-LAMP, RT-PCR and RT-qPCR detection of clinical samples

[0105]

[0106] In summary, the CAstV and ANV dual fluorescence RT-LAMP and RT-qPCR detection coincidence rates were 97.97% and 98.85%, respectively, indicating that the established method had a high coincidence rate and good consistency compared with RT-qPCR. The detection sensitivity was 10 times higher than that of ordinary RT-PCR, and the minimum detection limit was 10 2 The double fluorescence RT-LAMP method can save time and money, detect one or two pathogens in a single sample, and is suitable for use in low-income areas at the grassroots level, with the potential for large-scale on-site detection. This proves that with the continuous deepening of LAMP technology research, as well as the combination and optimization of other advanced technologies, more sensitive and portable diagnostic technology will provide strong support for the rapid diagnosis and effective prevention and control of diseases, which is of great significance for ensuring public health safety and promoting the development of the poultry industry.

[0107] Example 2

[0108] A dual fluorescence RT-LAMP detection kit for chicken astrovirus and avian nephritis virus comprises the following components: 2×Master Mix, Bst DNA polymerase, outer primers CAstV-F3, CAstV-B3, ANV-F3 and ANV-B3, loop primers CAstV-F3, CAstV-B3, ANV-F3 and ANV-B3, CAstV-Floop, CAstV-Bloop, ANV-Floop, ANV-Bloop, inner primers CAstV-BIP and ANV-BIP, FIP-FD and RNase-free Water; wherein FIP-FD comprises 0.48 μmol / L CAstV-FIP-FD and 0.32 μmol / L ANV-FIP-FD; CAstV-FIP-FD is a composite probe formed by annealing a probe CAstV-FIP and a probe CAstV-FD; and ANV-FIP-FD is a composite probe formed by annealing a probe ANV-FIP and a probe ANV-FD.

[0109] The reaction system of the dual fluorescence RT-LAMP detection kit is: 2×Master Mix 12.5μL, Bst DNA polymerase 0.1μL, outer primers CAstV-F3, CAstV-B3, ANV-F3 and ANV-B3 (working concentration 0.1μmol / L) 0.5μL each, loop primers CAstV-F3, CAstV-B3, ANV-F3 and ANV-B3 (working concentration 0.1μmol / L) 0.5μL each CAstV-Floop, CAstV-Bloop, ANV-Floop and ANV-Bloop (working concentration 0.2μmol / L) 0.5μL each, inner primers CAstV-BIP and ANV-BIP (working concentration 0.8μmol / L) 0.5μL each, FIP-FD addition amount is 0.5μL, DNA / cDNA template is 2μL, and RNase-free Water addition amount is 2.4μL.

[0110] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer and probe combination for dual fluorescent RT-LAMP detection of chicken astrovirus and avian nephritis virus, characterized in that: It includes primer CAstV-F3, primer CAstV-B3, probe CAstV-FIP, primer CAstV-BIP, primer CAstV-Floop, primer CAstV-Bloop, probe CAstV-FD, primer ANV-F3, primer ANV-B3, probe ANV-FIP, primer ANV-BIP, primer ANV-Floop, primer ANV-Bloop and probe ANV-FD, whose nucleotide sequences are shown as SEQ ID NO.1-14 respectively.

2. The primer and probe combination according to claim 1, characterized in that: The 3'-ends of the probe CAstV-FD ​​and the probe ANV-FD are labeled with different fluorescent groups; The 5'-ends of the probe CAstV-FIP and the probe ANV-FIP are labeled with different quenching groups.

3. The primer and probe combination according to claim 1, characterized in that: The 3' end of the probe CAstV-FD ​​is labeled with a FAM group; The 3' end of the probe ANV-FD is labeled with a CY3 group; The 5' end of the probe CAstV-FIP is labeled with a BHQ1 group; The 5' end of the probe ANV-FIP is labeled with a BHQ2 group.

4. A primer and probe combination for chicken astrovirus fluorescent RT-LAMP detection, characterized in that: It includes primer CAstV-F3, primer CAstV-B3, probe CAstV-FIP, primer CAstV-BIP, primer CAstV-Floop, primer CAstV-Bloop and probe CAstV-FD, whose nucleotide sequences are shown as SEQ ID NO.1-7 respectively.

5. The primer and probe combination according to claim 4, characterized in that: The 3' end of the probe CAstV-FD ​​is labeled with a fluorescent group; The 5' end of the probe CAstV-FIP is labeled with a quenching group.

6. A primer and probe combination for fluorescent RT-LAMP detection of avian nephritis virus, characterized in that: It includes primer ANV-F3, primer ANV-B3, probe ANV-FIP, primer ANV-BIP, primer ANV-Floop, primer ANV-Bloop and probe ANV-FD, whose nucleotide sequences are shown as SEQ ID NO.8-14 respectively.

7. The primer and probe combination according to claim 6, characterized in that: The 3' end of the probe ANV-FD is labeled with a fluorescent group; The 5' end of the probe ANV-FIP is labeled with a quenching group.

8. Use of the primer and probe combination according to any one of claims 1 to 3 in the preparation of a dual fluorescence RT-LAMP detection kit for chicken astrovirus and avian nephritis virus.

9. A dual fluorescence RT-LAMP detection kit for chicken astrovirus and avian nephritis virus, characterized in that: The method comprises the primer and probe combination according to any one of claims 1 to 3.

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