Primer group for identifying goose novel astrovirus genes type 1 and type 2 and application and kit thereof

Abstract

The invention belongs to the field of biology, and discloses a primer group for identifying goose novel astrovirus genes type 1 and type 2, and the primer group is a primer group for cutting an incision enzyme mediated isothermal amplification system. An upstream primer sequence and a downstream primer sequence in the primer group for identifying the novel goose astrovirus gene type 1 are as shown in SEQ ID NO.1 and SEQ ID NO.2; the sequences of upstream and downstream primers in the primer group for identifying the novel goose astrovirus gene type 2 are as shown in SEQ ID NO.3 and SEQ ID NO.4. The invention further discloses a method for identifying the novel goose astrovirus gene type 2. The primer group is developed aiming at a nicking endonuclease mediated isothermal amplification system, expensive detection equipment and reagents are not needed, the primer design is short, the pain points of other technologies are solved, and meanwhile, the difficulty that the primer group has serious side reaction and strict requirements on a reaction system and a detection method is overcome. Meanwhile, the invention further provides a kit based on the primer group and application of the kit.

Description

Technical Field

[0001] This invention relates to the field of biology, and more particularly to a primer set for identifying novel goose astrovirus genotypes 1 and 2, its application, and a kit. Background Technology

[0002] The novel goose astrovirus (N-GoAstV) primarily induces urate deposition in the joints and internal organs of 7-20 day old goslings, with death occurring 2-3 days after infection and peak mortality reaching 5-7 days. Surviving goslings become stunted, exhibiting severely impaired growth and development. Studies have also shown that the virus can be vertically transmitted through the reproductive tract, contaminating goose breeds. In recent years, various domestic scholars have discovered that the virus can also infect ducklings, Muscovy ducklings, and chicken flocks; the resulting economic losses are incalculable. Simultaneously, with the spread and mutation of the virus, different genotypes of N-GoAstV (N-GoAstV-1 and N-GoAstV-2) have been identified in the industry. Although N-GoAstV-1 (represented by strains such as AHDY and FLX) was the first to be reported, the predominantly circulating strains in the early stages of the epidemic were N-GoAstV-2 genotype strains (represented by strains such as HLJ01 and AHAU5). With the evolution of epidemiology, studies since 2022 have shown that the "dominant positive rate" of the N-GoAstV-2 genotype has gradually been replaced by the N-GoAstV-1 genotype, and many positive farms have experienced mixed infections of both N-GoAstV genotypes. Furthermore, mixed infections of N-GoAstV with goose parvovirus (GPV), goose reovirus (GREOV), and Riemerella anatipestifer (RA) further complicate clinical diagnosis. Currently, there are no effective drugs or biological agents for the prevention and treatment of this disease in clinical practice, making the establishment of clinically suitable diagnostic methods crucial for its control. Therefore, establishing a simple, accurate, and rapid dual diagnostic method for N-GoAstV-1 / 2 is urgently needed.

[0003] Because the common symptom of N-GoAstV infection is urate deposition in multiple organs and joints (gout syndrome), it is difficult to distinguish from non-pathogen-induced gout syndrome, and differentiation is difficult based solely on clinical and necropsy observations. Current laboratory diagnostic methods for N-GoAstV-1 / 2 mainly include isolation and culture identification, serological diagnosis, and molecular biological methods. Among these, isolation and culture are time-consuming, which is not conducive to disease control and epidemic management. Established serological diagnostic methods for GoAstV mainly include enzyme-linked immunosorbent assay (ELISA), immunochromatographic strip assay (ICS), and photoelectrochemical immunosensor (PEC-IS). ELISA is a sensitive and accurate serological immunological diagnostic technique, but its operation is relatively complex and the detection process is time-consuming; ICS and PEC-IS have short and easy-to-operate procedures, but their low sensitivity limits their clinical application. In addition, the antibodies used in serological diagnostic methods may be incompatible with N-GoAstV-1 and N-GoAstV-2 or have varying degrees of cross-reactivity, which affects the accuracy of these methods and poses a greater challenge in distinguishing between the two N-GoAstV genotypes.

[0004] In recent years, with the development of molecular biology-related technologies, nucleic acid amplification has become the most core and commonly used method in gene diagnostic technology. PCR and PCR-derived technologies have been increasingly widely used and developed for the diagnosis of N-GoAstV. However, these methods have the following shortcomings: (1) They require expensive PCR instruments and are relatively complicated to operate, which increases the cost of testing at the grassroots level; (2) Polymerase is sensitive to various body fluid components, and nucleic acid extraction kits must be used to obtain "high-quality" nucleic acid templates; (3) They require precise temperature cycling processes, otherwise nucleic acid amplification will be affected; (4) The amplification reaction time is long. When ordinary PCR detects RNA viruses such as N-GoAstV, a reverse transcription process needs to be added in advance, plus complex denaturation, annealing and extension temperature cycling processes, which generally take several hours. In addition, electrophoresis and imaging processes further prolong the detection time, making it difficult to promote and apply at the grassroots level.

[0005] Regarding the identification techniques for goose astrovirus genotypes 1 and 2, see the patent application with publication number CN116103441A, which is entitled "Multiplex fluorescent PCR primer and probe set, method and application for detecting goose astrovirus genotypes 1 and 2". It uses fluorescent PCR to identify these two genotypes. As mentioned above, due to the difficulties in detection equipment and cost, there is a need to develop new detection technologies.

[0006] Isothermal nucleic acid amplification technology can at least partially solve the above problems. Isothermal nucleic acid amplification technologies include strand displacement amplification (SDA), loop-mediated isothermal nucleic acid amplification (LAMP), nucleic acid sequence amplification (NASBA), and rolling circle amplification (RCA). Taking SDA as an example, it requires the use of thio-modified dNTPs, which are expensive.

[0007] Another branch of nucleic acid isothermal amplification technology is nicking endonuclease-mediated isothermal amplification (NEAR). The nicking endonuclease in this method is a special type II restriction enzyme. Unlike ordinary restriction enzymes, it does not cut double-stranded DNA (dsDNA), but only cuts a specific strand of the DNA double helix. However, NEAR also has obvious disadvantages: it is prone to serious side reactions, and most nicking endonucleases only recognize specific 4-8 bp sequences. If there is no corresponding site on the template, it cannot be used.

[0008] Therefore, how to easily, quickly, and accurately identify goose astrovirus genotypes 1 and 2 is a difficult problem. Summary of the Invention

[0009] The purpose of this invention is to provide a primer set for identifying goose astrovirus genotypes 1 and 2. This primer set is developed for nicking endonuclease-mediated isothermal amplification systems. It not only eliminates the need for expensive detection equipment and reagents, but also features a relatively short primer design, thus solving the pain points of other technologies. At the same time, it overcomes the difficulties of severe side reactions and stringent requirements on reaction systems and detection methods.

[0010] In addition, the present invention also provides a kit based on this primer set and its applications.

[0011] To achieve the above objectives, this application discloses a primer set for identifying goose novel astrovirus genotypes 1 and 2, wherein the primer set is a primer set for nicking endonuclease-mediated isothermal amplification system;

[0012] The upstream and downstream primer sequences in the primer set used to identify the novel goose astrovirus genotype 1 are shown in SEQ ID NO.1 and SEQ ID NO.2;

[0013] The upstream and downstream primer sequences in the primer set used to identify the novel goose astrovirus genotype 2 are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0014] Nicking endonuclease-mediated isothermal amplification (NEAR) is a relatively unstudied isothermal nucleic acid amplification technique, developed by researchers at Ionian Technologies in 2008. In addition to the strand displacement enzyme (Bst), a nicking endonuclease is added to the NEAR reaction. Primer design for the NEAR reaction requires adding the DNA sequence of the nicking endonuclease to the 5' end of the primer along with a "stable sequence." The overall reaction can be divided into two steps. In the first step, Bst reacts with the template DNA to generate a small amount of DNA containing the nicking endonuclease recognition sequence. This DNA serves as a template in the second step, where the nicking endonuclease first cleaves the single strand, and then Bst synthesizes new strands. Essentially, a large amount of newly synthesized DNA can be formed within 5-10 minutes for detection.

[0015] Its advantages are: rapid reaction, low equipment requirements, and short primers;

[0016] Its disadvantages are equally obvious: it is extremely prone to serious side reactions and has stringent requirements for the reaction system and detection methods.

[0017] Through repeated screening and research on the relevant gene fragments of these two N-GoAstV genotypes, the present invention has finally developed the primer set as described above. When applied to the nicking endonuclease-mediated isothermal amplification system, this primer set has the advantages of no side reactions and clear bands.

[0018] In some preferred embodiments of the present invention, a kit was obtained by studying the buffer system, which is simple to operate and has reduced system requirements when used in the nicking restriction enzyme-mediated isothermal amplification method.

[0019] Meanwhile, the present invention also discloses the use of a detection kit prepared using the primer set described above; the detection kit is used to identify goose novel astrovirus genotypes 1 and 2; the kit is a kit based on nicking endonuclease-mediated isothermal amplification technology.

[0020] In addition, the present invention also discloses a kit based on nicking endonuclease-mediated isothermal amplification technology, containing the primer set as described above.

[0021] In the above-described kit, the kit contains a buffer solution; the buffer solution is a phosphate buffer solution with a pH of 7.4.

[0022] In the above-mentioned kit, the kit contains polymerase and nicking enzyme; the polymerase is Bst DNA3.0 polymerase; the nicking enzyme is WarmStart® Nt.BstNBI nicking enzyme.

[0023] The present invention has the following advantages and effects compared with the prior art:

[0024] Based on the newly discovered N-GoAstV-1 and N-GoAstV-2 specific nucleic acid sequences in recent years, this invention applies a dual isothermal nucleic acid detection method mediated by a hot-start nicking enzyme (WarmStart® Nt.BstNBI nicking enzyme) and a strand substitution polymerase (BstDNA3.0 polymerase). Utilizing the reverse transcription properties of BstDNA3.0 polymerase and the nicking properties of WarmStart® Nt.BstNBI nicking enzyme, the additional reverse transcription and temperature cycling processes of conventional RT-PCR are eliminated, significantly shortening the reaction process. This invention can be used simultaneously for the diagnosis of infectious gout syndrome in goslings caused by both N-GoAstV-1 and N-GoAstV-2.

[0025] The primers and reagents of this invention have high specificity and sensitivity, and are simple and quick to operate, making them very suitable for the diagnosis, screening and prevention of N-GoAstV-1 and N-GoAstV-2 in primary laboratories. Attached Figure Description

[0026] Figure 1 Agarose gel images showing amplification results for two genotypes using five primer pairs with different "stable regions";

[0027] Figure 2 Agarose gel images of primer pairs selected for two genotypes and their results for N-GoAstV positive nucleic acids of different genotypes;

[0028] Figure 3 The amplification results of dual NEAR in reaction buffers at different pH values ​​are shown in the figure.

[0029] Figure 4 The amplification results of dual NEAR at different reaction temperatures are shown in the figure.

[0030] Figure 5 The amplification results of dual NEAR at different reaction times are shown in the figure.

[0031] Figure 6 Agarose gel images showing the amplified results of two genotypes of N-GoAstV-ORF1b using standard plasmid templates and compared with PCR.

[0032] Figure 7 Agarose gel image showing the results of dual NEAR specific detection. Detailed Implementation

[0033] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0034] 1.1 Screening for optimal primer pairs and "stable region" sequences

[0035] Unlike conventional isothermal diagnostic methods, the 5' end of the nicking primers needs to be sequentially supplemented with a "stable region" and the corresponding restriction enzyme site. The sequences in the primers of this invention that bind to the target DNA are designed using Primer Premier 5.0 targeting the ORF1b gene region of N-GoAstV-1 / 2 (N-GoAstV-1 was designed with reference to the TZ03 strain (gene search number: MW353015), and N-GoAstV-2 was designed with reference to the SD03 strain (gene search number: OP621341)).

[0036] For the "stable region" sequences of the nicked primers, this invention designed 43 candidate "stable region" sequences, including "TGGATCTGAT", "GTTATCGTAACT", and "TCGTTCTGCT". The designed "stable region" sequences were evaluated using the OligoAnalyzer™: Primer analysis tool in conjunction with the primers, and preliminary practical validation experiments were conducted. Only 7 "stable region" sequences were suitable for the designed primer set. Finally, this invention, combined with preliminary experiments, screened out the "stable region" sequences... CTATATTCAC "The combined primer pairs produce bright NEAR amplification bands that are less prone to nonspecific amplification."

[0037] To further evaluate the impact of the "stable region" on amplification results and to screen the optimal primer set, this invention compares the reference "stable region" sequences in existing patents and published literature: Reference "stable region" sequence 1 "Research on Nucleic Acid Amplification and Detection Technology for Food_Wang Liu, Doctoral Dissertation of Zhejiang University", and Reference "stable region" sequence 2 "CN103173537B_Rapid Detection Kit for Isothermal Amplification of Nucleic Acid with Nucleotide Endonuclease of Escherichia coli O157_3AH7".

[0038] All primer sequences are shown in Tables 1 and 2 (the italicized “GAGTC” indicates the Nt.BstNBI nicking enzyme site, and the underlined sequence indicates the “stable region”).

[0039] Table 1 Primers required for the N-GoAstV-1-NEAR reaction

[0040] Table 2 Primers required for the N-GoAstV-2-NEAR reaction

[0041] RNA extraction from test samples: Liver, spleen, and kidney tissues from virus-infected goslings were homogenized. 20-30 mg of the homogenate was added to 100-200 μL of deionized water as the test sample. 20 μL of the test sample was added to 5 μL of nucleic acid release agent (ultra-rapid nucleic acid release agent (universal type), purchased from Weifang Anpu Future Biotechnology Co., Ltd., for extracting tissue RNA) and gently mixed into a 200 μL nuclease-free PCR tube. The PCR tube containing the mixed sample solution was placed in a metal / water bath and incubated at 95℃ for 5 min. The sample was then removed and allowed to equilibrate at room temperature for 3 min, followed by centrifugation at 10000 rpm for 2 min at room temperature. The supernatant was directly used for subsequent amplification procedures.

[0042] In the screening of "stable regions" and nicking primer assays, isothermal amplification (NEAR) of the nicking endonuclease was performed using Bst DNA 3.0 polymerase and WarmStart® Nt.BstNBI nicking enzyme manufactured by New England Biolabs, Inc. (NEB), along with the reaction buffer (NEBuffer™ r3.1) provided for both enzymes. The reaction conditions were 65°C as recommended by NEB for both enzymes, and the reaction time was 1 h. The reaction system used was as follows:

[0043] NEBuffer™ r3.1 (10×) 2.0 μL

[0044] Upstream nicking primer (10 μM) 2.0 μL

[0045] Downstream nicking primer (10 μM) 2.0 μL

[0046] dNTPs (final concentration 1.5 mM) 4.4 μL

[0047] 0.5 μL of Bst DNA 3.0 polymerase

[0048] WarmStart® Nt.BstNBI 0.1 μL

[0049] Template RNA or positive control (concentration 10 ng / μL) 1 μL

[0050] To bring the DEPC water level up to 20 μL.

[0051] For detailed results, please refer to... Figure 1 ; Figure 1 The images show the agarose gel amplification results for two genotypes using five primer pairs with different "stable regions". Figure 1 In the diagram, A1, B1, and C1 represent the "stable region" used in this invention for the N-GoAstV-1 type. Referring to "stable regions" 1 and 2, the amplification results of different primer pairs are shown. Lanes 1, 2, 3, 4, and 5 represent the amplification results of primer pairs FN11 / RN11, FN12 / RN12, FN13 / RN13, FN14 / RN14, and FN15 / RN15, respectively. Lanes 6, 7, 8, 9, and 10 represent the amplification results of the negative controls corresponding to primer pairs FN11 / RN11, FN12 / RN12, FN13 / RN13, FN14 / RN14, and FN15 / RN15, respectively. Figure 1 In the diagram, A2, B2, and C2 represent the stable regions used in this invention, specifically the N-GoAstV-2 type. Referencing stable regions 1 and 2, the amplification results are shown using different primer pairs. Lanes 1, 2, 3, 4, and 5 represent the amplification results for primer pairs FN21 / RN21, FN22 / RN22, FN23 / RN23, FN24 / RN24, and FN25 / RN25, respectively. Lanes 6, 7, 8, 9, and 10 represent the negative control amplification results for primer pairs FN21 / RN21, FN22 / RN22, FN23 / RN23, FN24 / RN24, and FN25 / RN25, respectively. M represents the DNA molecular weight standard DL2000.

[0052] Gel electrophoresis imaging showed that the bands amplified by N-GoAstV-1 primers (FN12 / RN12 and FN13 / RN13) and N-GoAstV-2 primers (FN21 / RN21, FN23 / RN23 and FN24 / RN24) paired with "stable region" 1 were all weak, indicating that they were not suitable for the detection of N-GoAstV-1 / 2. On the other hand, non-specific product bands appeared by N-GoAstV-1 primers (FN11 / RN11 and FN12 / RN12) and N-GoAstV-2 primers (FN23 / RN23 and FN24 / RN24) paired with "stable region" 2, indicating that using different "stable region" sequences upstream and downstream can easily increase the risk of non-specific amplification. The N-GoAstV-1 type primers (FN12 / RN12 and FN13 / RN13) and N-GoAstV-2 type primers (FN21 / RN21, FN23 / RN23 and FN24 / RN24) paired with the "stable region" used in this invention all amplified a single target band, but the FN24 / RN24 primer pair amplified a non-specific band, so it was screened out.

[0053] 1.2 Detection of "crossover" between genotypes using primer pairs of different genotypes

[0054] The N-GoAstV-1 type primers (FN12 / RN12 and FN13 / RN13) and N-GoAstV-2 type primers (FN21 / RN21 and FN23 / RN23) paired with the "stable region" used in this invention were selected. The above reaction system was used to detect N-GoAstV-1 and N-GoAstV-2 positive nucleic acids respectively for screening through "cross-testing" between genotypes.

[0055] Figure 2 Agarose gel images showing the results of primer pairs selected for two genotypes for detecting N-GoAstV positive nucleic acids of different genotypes. Figure 2 In the diagram, A represents the amplification results of four primer pairs detecting N-GoAstV-1 positive nucleic acid. Lanes 1, 2, 3, and 4 represent the amplification results of primer pairs FN12 / RN12, FN13 / RN13, FN21 / RN21, and FN23 / RN23, respectively. Lanes 5, 6, 7, and 8 represent the amplification results of the negative controls corresponding to primer pairs FN12 / RN12, FN13 / RN13, FN21 / RN21, and FN23 / RN23, respectively. Figure 2 In the diagram, B represents the amplification results of four primer pairs detecting N-GoAstV-2 positive nucleic acid. Lanes 1, 2, 3, and 4 represent the amplification results of primer pairs FN12 / RN12, FN13 / RN13, FN21 / RN21, and FN23 / RN23, respectively. Lanes 5, 6, 7, and 8 represent the amplification results of the negative controls corresponding to primer pairs FN12 / RN12, FN13 / RN13, FN21 / RN21, and FN23 / RN23, respectively. M represents the DNA molecular weight standard DL2000.

[0056] Gel electrophoresis imaging Figure 2 Figure A indicates that primer pairs FN12 / RN12 and FN13 / RN13 can only detect N-GoAstV-1 positive nucleic acids, consistent with expectations. However, the amplified band of primer pair FN12 / RN12 is significantly brighter than that of FN13 / RN13. To ensure the sensitivity of the detection results, primer pair FN13 / RN13 was removed, and primer pair FN12 / RN12 was retained for downstream detection of N-GoAstV-1 positive nucleic acids. Gel electrophoresis imaging. Figure 2 B in the diagram indicates that primer pairs FN21 / RN21 and FN23 / RN23 can detect N-GoAstV-2 positive nucleic acids, which is consistent with expectations. However, primer pair FN21 / RN21 showed non-specific bands for N-GoAstV-1 positive nucleic acids. To ensure the accuracy and specificity of the detection results, primer pair FN21 / RN21 was removed, and primer pair FN23 / RN23 was retained for downstream detection of N-GoAstV-2 positive nucleic acids.

[0057] Ultimately, this invention selected FN12 / RN12 and FN23 / RN23 to establish a dual NEAR reaction for subsequent experiments.

[0058] 1.3 Screening for the optimal reaction buffer for dual NEAR

[0059] Because the Tris-HCl buffer system used in the reaction buffer (NEBuffer™ r3.1) provided by New England Biolabs, Inc. decreases the pH of the reaction solution as the reaction temperature increases, thus affecting the amplification efficiency of the two enzymes, this invention selected phosphate buffer systems with different pH values ​​for reaction experiments and compared them with NEBuffer™ r3.1. The reaction temperature was 65°C, and the reaction time was 1 h. The formulations of the prepared buffer systems are shown in Table 3:

[0060] Table 3. Buffer system formulation of the present invention (100 mL, 10×)

[0061] The dual NEAR reaction system used is as follows:

[0062] Self-prepared buffer solution (10×) 2.0 μL

[0063] 2.0 μL each of primer pair FN12 / RN12 (10 μM)

[0064] 2.0 μL each of primer pair FN23 / RN23 (10 μM)

[0065] dNTPs (final concentration 1.5 mM) 4.4 μL

[0066] 0.5 μL of Bst DNA 3.0 polymerase

[0067] WarmStart® Nt.BstNBI 0.1 μL

[0068] Template RNA or positive control (concentration 10 ng / μL) 1 μL

[0069] To bring the DEPC water level up to 20 μL.

[0070] Figure 3This image shows the amplification results of double NEAR in reaction buffers at different pH values. The template used was N-GoAstV-1 / 2 double-positive nucleic acid. Lanes 1, 2, 3, 4, 5, 6, 7, 8, and 9 show the amplification results in self-prepared buffers at pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, and pH 7.9, respectively. Lane 10 shows the amplification results using NEBuffer™ r3.1, and lane 11 is the negative control. M represents the DNA molecular weight standard DL2000.

[0071] Gel electrophoresis imaging Figure 3 The results showed that the amplification effect was best when using a self-prepared buffer at pH 7.4. Therefore, the optimal reaction temperature was explored by using a self-prepared 10× buffer (KH2PO4 160 mM, Na2HPO4 640 mM, 60 mM MgCl2, 800 µg / mL recombinant albumin).

[0072] 1.4 Optimal reaction temperature for dual NEAR

[0073] After determining the NEAR reaction system, the optimal reaction temperature for N-GoAstV dual NEAR was explored. The temperature gradient was set at 61~69℃, the template used was N-GoAstV-1 / 2 double-positive nucleic acid, and the reaction time was 1 h.

[0074] Figure 4 The images show the amplification results of the double NEAR at different reaction temperatures. Lanes 1, 2, 3, 4, 5, 6, 7, 8, and 9 represent the amplification results at 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, and 69 ℃, respectively. Lane 10 is the negative control. M represents the DNA molecular weight standard DL2000.

[0075] Gel electrophoresis imaging Figure 4 This indicates that the dual NEAR can amplify the target band at all temperatures, but the band signal intensity at 66 ℃ is significantly higher than that of other groups. Furthermore, the difference between lanes at 65 ℃ and 67 ℃ and lanes at 66 ℃ is not significant. Therefore, 66 ℃ is determined to be the optimal reaction temperature. Moreover, the N-GoAstV dual NEAR established in this invention is a "wide-temperature" reaction, highlighting that this technology can achieve good amplification results even under less precise isothermal conditions, which is beneficial for its promotion and application in grassroots laboratories.

[0076] 1.5 Dual NEAR Optimal Response Time

[0077] After determining the optimal reaction temperature, the optimal reaction time was explored. The temperature used was 66℃, the template used was N-GoAstV-1 / 2 double-positive nucleic acid, and the reaction was set with an amplification time gradient of 20~40 min (5 min interval).

[0078] Figure 5 The images show the amplification results of the double NEAR at different time points. Lanes 1, 2, 3, 4, and 5 represent the amplification results at reaction times of 20 min, 25 min, 30 min, 35 min, and 40 min, respectively. Lane 6 is the negative control. M represents the DNA molecular weight standard DL2000.

[0079] The results showed that the target band could be detected at a reaction time of 20 minutes, and the brightness of the target band reached its peak at a reaction time of 35 minutes. Therefore, a reaction time of 35 minutes was selected as the optimal reaction time.

[0080] 1.6 Dual NEAR Sensitivity Assessment

[0081] The standard plasmid templates containing the full-length sequences of N-GoAstV-1-ORF1b and N-GoAstV-2-ORF1b at the determined concentrations were serially diluted 10-fold. The detection sensitivity of the two methods was compared by double NEAR with optimized conditions (reaction at 66℃ for 35 min) and conventional PCR (primers used were from the published literature (Epidemiological Investigation of Goose Astrovirus in Hebei Province, China, 2019-2021)).

[0082] Figure 6 The images show the agarose gel images of the amplified nucleic acids of the two genotypes compared using double NEAR and PCR, respectively. Figure 6 A1 and A2 in the image show the amplification results of the N-GoAstV-1 standard plasmid using PCR and NEAR, respectively. Lanes 1, 2, 3, 4, 5, 6, and 7 represent the template amount of the N-GoAstV-1 standard plasmid, which is 1.48 × 10⁻⁶. 5 ~1.48×10 -1 Copy, and dilute 10 times in sequence, with lane 8 as the negative control. Figure 6 In the image, B1 and B2 represent the amplification results of the N-GoAstV-2 standard plasmid using PCR and NEAR, respectively. Lanes 1, 2, 3, 4, 5, 6, and 7 show the template amount of the N-GoAstV-2 standard plasmid, which is 7.13 × 10⁻⁶. 5 ~7.13×10 -1Copy, then dilute 10-fold sequentially; lane 8 serves as the negative control. M represents the DNA molecular weight standard DL2000.

[0083] like Figure 6 As shown, the dual NEAR method established in this invention can still amplify the target band when the N-GoAstV-1 template concentration is reduced to 14.8 copies / μL, with sensitivity consistent with the PCR method; when the N-GoAstV-2 template concentration is reduced to 7.13 copies / μL, it can still amplify the target band, with sensitivity 10 times higher than PCR. This indicates that the dual NEAR method established in this study has extremely high sensitivity for detecting both N-GoAstV-1 and N-GoAstV-2.

[0084] 1.7 Dual NEAR-Specific Assessment

[0085] The dual NEAR system established in this invention was used to detect N-GoAstV-1, N-GoAstV-2, and N-GoAstV-1 / 2 double-positive nucleic acids, and four control pathogens—goose parvovirus (GPV), goose circovirus (GoCV), goose reovirus (GREOV), and tembusuvirus (TMUV)—were also tested.

[0086] Figure 7 This is an agarose gel image showing the results of dual NEAR specific detection. Lanes 1, 2, 3, 4, 5, 6, and 7 represent the detection results for N-GoAstV-1 positive nucleic acid, N-GoAstV-2 positive nucleic acid, N-GoAstV-1 / 2 double positive nucleic acid, GPV positive nucleic acid, GoCV positive nucleic acid, GREOV positive nucleic acid, and TMUV positive nucleic acid, respectively. Lane 8 is the negative control. M represents the DNA molecular weight standard DL2000.

[0087] like Figure 7 As shown, both N-GoAstV-1 and N-GoAstV-2 positive nucleic acids exhibited single-target band amplification, and N-GoAstV-1 / 2 double-positive nucleic acids showed double-entry band amplification. No amplification bands were observed in the other virus detection lanes. Three independent replicate experiments demonstrate that the dual NEAR system established in this invention has high specificity for the detection of N-GoAstV and shows no cross-reactivity with other pathogens.

[0088] 1.8 Results of dual NEAR clinical sample testing

[0089] To further evaluate the accuracy of the dual NEAR method for N-GoAstV detection, RT-PCR and the dual NEAR method were used to detect N-GoAstV in collected clinical samples. Statistical analysis was performed using the Kappa (κ) test in SPSS 24.0 software to compare the concordance between the two methods and to comprehensively evaluate the superiority of the N-GoAstV-NEAR method for detecting clinical samples.

[0090] Thirty-five samples that were strongly positive by RT-PCR and had correct sequencing results (14 positive for N-GoAstV-1 and 21 positive for N-GoAstV-2) were selected for positive verification. The results showed that the double NEAR detection rate was 100%, which was completely consistent with the RT-PCR method.

[0091] Parallel testing of RT-PCR and double NEAR was performed on 379 collected clinical samples. The positive detection rate of RT-PCR for N-GoAstV-1 clinical positive samples was 18.73% (71 / 379), and the positive detection rate of double NEAR was 19.00% (72 / 379). The positive detection rate of both methods for N-GoAstV-2 clinical positive samples was 30.34% (115 / 379). The positive detection rate of RT-PCR for N-GoAstV-1 / 2 clinical double positive samples was 6.07% (23 / 379), and the positive detection rate of double NEAR was 5.54% (21 / 379). Statistical results showed that the two detection methods were highly consistent, as shown in Table 4. The accuracy of the double NEAR method was verified by subsequent virus isolation and sequencing analysis of one clinically positive N-GoAstV-1 sample and two clinically double positive N-GoAstV-1 / 2 samples that were missed by RT-PCR. These results also further demonstrate that the double NEAR method has higher sensitivity than conventional RT-PCR.

[0092] Table 4 Comparative analysis of the results of dual NEAR and RT-PCR detection

[0093] Results analysis:

[0094] 1. As can be seen from the test in Section 1.1 of this invention, in the nicking endonuclease-mediated isothermal amplification technology, the selection of the site and the design of the "stable region" are two very important key technical parameters. The two together determine whether there are side reactions and whether there will be cross-detection errors.

[0095] This invention not only screens a large number of stable regions, but also verifies the application effect of mature and stable "stable regions" in the field in the detection of novel goose astroviruses, proving that stable regions play a core role in the detection of this invention.

[0096] Meanwhile, the choice of sites demonstrates that not just any site can achieve the purpose of this invention. This is a significant difference between nicking endonuclease-mediated isothermal amplification technology and traditional PCR or fluorescent PCR; in traditional technologies, as long as the site is conserved, the detection objective can generally be achieved. It is precisely because of the inherent difficulty in developing nicking endonuclease-mediated isothermal amplification technology that the development workload is enormous.

[0097] However, we must also recognize the benefits of the successful development of nicking endonuclease-mediated isothermal amplification technology, which reduces the requirements for equipment and the difficulty of personnel operation compared to traditional technologies.

[0098] 2. Section 1.2 of this invention demonstrates that even stable primer design has a significant antagonistic effect in nicking endonuclease-mediated isothermal amplification technology. Therefore, developing stable primers is only the first step in realizing nicking endonuclease-mediated isothermal amplification technology. Developing double or multiple nicking endonuclease-mediated isothermal amplification technology obviously increases the difficulty.

[0099] 3. Section 1.3 of this invention abandons the mature Tris-HCl buffer system and adopts a self-developed phosphate buffer system, and verifies the optimal pH reaction conditions, thus realizing the accurate detection of this invention.

[0100] 4. Sections 1.4 to 1.8 of this invention demonstrate the advantages of this invention from multiple perspectives, including other process parameters, detection specificity, and sensitivity.

[0101] In summary, this study successfully developed a nicking endonuclease-mediated isothermal amplification protocol for the differentiation of N-GoAstV-1 and N-GoAstV-2 types. This protocol is highly specific and sensitive, and is simple and quick to operate, making it very suitable for the diagnosis, screening, and prevention of N-GoAstV-1 and N-GoAstV-2 types in primary care laboratories.

Claims

1. A primer set for identifying goose novel astrovirus genotypes 1 and 2, characterized in that, The primer set is used for nicking the endonuclease-mediated isothermal amplification system; The upstream and downstream primer sequences in the primer set used to identify goose novel astrovirus genotype 1 are shown in SEQ ID NO.1 and SEQ ID NO.2; The upstream and downstream primer sequences in the primer set used to identify goose astrovirus genotype 2 are shown in SEQ ID NO.3 and SEQ ID NO.

4.

2. The use of the detection kit prepared using the primer set as described in claim 1; the detection kit is used to identify goose novel astrovirus genotypes 1 and 2; the kit is a kit based on nicking endonuclease-mediated isothermal amplification technology.

3. A kit based on nicking endonuclease-mediated isothermal amplification technology, characterized in that, It contains the primer set as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The kit contains a buffer solution; the buffer solution is a compound phosphate buffer with a pH of 7.

4.

5. The reagent kit according to claim 3, characterized in that, The kit contains a polymerase and a nicking enzyme; the polymerase is Bst DNA3.0 polymerase; the nicking enzyme is WarmStart® Nt.BstNBI nicking enzyme.

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