Dual LAMP (loop-mediated isothermal amplification) detection system as well as kit and detection method thereof

Abstract

The invention relates to a dual LAMP (loop-mediated isothermal amplification) detection system as well as a kit and a detection method thereof. The dual LAMP detection system comprises a primer group, wherein the primer group comprises four primers for specifically detecting the cap gene of the duck circovirus and four primers for specifically detecting the Fiber2 gene of the duck adenovirus type 3. The invention provides a duck circovirus and duck adenovirus type 3 dual LAMP detection system which is efficient, accurate and easy to operate and a corresponding kit. The kit does not need to be operated by complex equipment or professionals, can detect the duck circovirus and the duck adenovirus type 3 at the same time, can introduce a visual detection technology, is suitable for grassroots veterinary laboratory and on-site rapid detection, provides a new detection method for the duck circovirus and the duck adenovirus type 3 for grassroots veterinarians and farmers, and can be used for detecting the duck circovirus and the duck adenovirus type 3 at the same time. Therefore, the requirement on rapid diagnosis of the two viruses in practical application is met.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a dual LAMP detection system and a kit and a detection method thereof. Background Art

[0002] Duck circovirus (DuCV) and duck adenovirus type 3 (DAdV-3) are two major pathogens in waterfowl farming. Each has unique biological characteristics and causes serious economic losses to the duck farming industry. Duck circovirus is a small, non-enveloped, single-stranded DNA virus belonging to the Circoviridae family. Its genome structure is complex and encodes multiple proteins, some of which have functions such as regulating the host cell cycle and inducing cell apoptosis. Duck circovirus infection can cause immunosuppression in ducks, resulting in decreased body resistance and susceptibility to other pathogens. In addition, the virus can also infect offspring ducklings through vertical transmission, causing a more widespread epidemic. Duck adenovirus type 3 belongs to the Adenoviridae family and is an enveloped double-stranded DNA virus. Infection with this virus can cause serious diseases such as hepatitis and hemorrhagic enteritis in ducks. In recent years, the infection rate and incidence of DAdV-3 in the duck farming industry have been on the rise, and it has become one of the important epidemic diseases in the duck farming industry. DAdV-3 infection is highly contagious in ducks and can lead to large-scale outbreaks, causing serious economic losses to the duck farming industry. Given the harmfulness and transmission characteristics of these two viruses, it is particularly important to establish a fast, accurate and simple detection method.

[0003] At present, the detection methods for duck circovirus and duck adenovirus type 3 mainly include electron microscopy, ELISA, PCR, qPCR, etc., but these methods require complex experimental equipment and strict experimental conditions, which are not convenient for rapid on-site detection. As an emerging nucleic acid amplification technology, loop-mediated isothermal amplification (LAMP) technology is based on the principle of designing four primers for six regions of the target gene, and using Bst chain displacement DNA polymerase to specifically and efficiently amplify DNA fragments under constant temperature conditions. However, there has been no report on the application of LAMP technology to the dual detection of duck circovirus and duck adenovirus type 3, probably due to the easy interference between primers and the difficulty in optimizing the conditions of multiple LAMP detection. Summary of the invention

[0004] The purpose of the present invention is to disclose a dual LAMP detection system and a kit and a detection method thereof, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions: The first aspect of the present invention is to provide a dual LAMP detection system for simultaneously amplifying duck circovirus and duck adenovirus type 3.

[0006] The second aspect of the present invention is to provide a kit comprising the dual LAMP detection system described in the first aspect of the present invention.

[0007] The third aspect of the present invention is to provide a detection method for the kit described in the second aspect of the present invention for non-disease diagnosis purposes.

[0008] The dual LAMP detection system described in the first aspect of the present invention includes a primer set, which includes primers for specifically detecting the duck circovirus cap gene: DuCV-F3, DuCV-B3, DuCV-FIP and DuCV-BIP; and primers for specifically detecting the duck adenovirus type 3 Fiber2 gene: DAdV-3-F3, DAdV-3-B3, DAdV-3-FIP and DAdV-3-BIP.

[0009] Duck circovirus cap protein (GenBank accession number NC_007220.1) is the main component of duck circovirus capsid and has the function of immune protective antigen. Duck adenovirus type 3 fiber 2 protein (GenBank accession number OM201326.1) is an important structural protein on the surface of duck adenovirus, with subgroup and type specific epitopes, which can induce the production of neutralizing antibodies and bind to cell receptors, play a key role in the early stage of viral infection, and have high conservation among different duck adenovirus type 3 strains. The dual LAMP detection system amplifies the conserved sites of the important gene species of these two viruses, and can efficiently, accurately and conveniently obtain the corresponding amplification results to meet the needs of rapid diagnosis of these two viruses in practical applications.

[0010] In some embodiments of the first aspect of the present invention, the nucleotide sequence of the DuCV-F3 is shown in SEQ ID No:5; the nucleotide sequence of the DuCV-B3 is shown in SEQ ID No:6; the nucleotide sequence of the DuCV-FIP is shown in SEQ ID No:7; the nucleotide sequence of the DuCV-BIP is shown in SEQ ID No:8; the nucleotide sequence of the DAdV-3-F3 is shown in SEQ ID No:13; the nucleotide sequence of the DAdV-3-B3 is shown in SEQ ID No:14; the nucleotide sequence of the DAdV-3-FIP is shown in SEQ ID No:15; the nucleotide sequence of the DAdV-3-BIP is shown in SEQ ID No:16.

[0011] In some embodiments of the first aspect of the present invention, the molar ratio of the DuCV-F3, the DuCV-B3, the DuCV-FIP and the DuCV-BIP is 1:1:4:4.

[0012] In some embodiments of the first aspect of the present invention, the molar ratio of the DAdV-3-F3, the DAdV-3-B3, the DAdV-3-FIP and the DAdV-3-BIP is 1:1:4:4.

[0013] In some embodiments of the first aspect of the present invention, the dual LAMP detection system further comprises a loop-mediated isothermal amplification buffer.

[0014] The duck circovirus and duck adenovirus type 3 dual detection kit described in the second aspect of the present invention comprises the above-mentioned dual LAMP detection system.

[0015] In some embodiments of the second aspect of the present invention, the kit further comprises at least one of a Gelgreen colorimetric solution, a positive control quality control product, and a negative control quality control product. The Gelgreen colorimetric solution is directly added to the amplification product, so that the user can directly know whether the test sample contains duck circovirus or duck adenovirus type 3 by color change.

[0016] In some embodiments of the second aspect of the present invention, the positive control product is a mixture of a plasmid containing the duck circovirus cap gene and a plasmid containing the duck adenovirus type 3 Fiber2 gene.

[0017] In some embodiments of the second aspect of the present invention, the dual LAMP detection system is 25uL, including loop-mediated isothermal amplification buffer 2× LAMP Master Mix 12.5 μL (1×), outer primer DuCV-F3 0.25 μL (20 μmol / L), outer primer DAdV-3-F3 0.25 μL (20 μmol / L), outer primer DuCV-B3 0.25 μL (20 μmol / L), outer primer DAdV-3-B3 0.25 μL (20 μmol / L), inner primer DuCV-FIP 1 μL (20 μmol / L), inner primer DAdV-3-FIP 1 μL (20 μmol / L), inner primer DuCV-BIP 1 μL (20 μmol / L), inner primer DAdV-3-BIP 1 μL (20 μmol / L), positive control quality control product 2 μL, Bst polymerase 1 μL (20 μmol / L), and 1 μL of PCR amplification. μL and 4.5 μL ddH2O.

[0018] The detection method according to the third aspect of the present invention comprises the following steps: (1) Extract DNA from the sample to be tested as a reaction template; (2) Mixing the primer set, the loop-mediated isothermal amplification buffer and the reaction template to perform a loop-mediated isothermal amplification reaction.

[0019] After the amplification reaction is completed, the amplification product is subjected to 1.5% agarose gel electrophoresis to determine whether there is a band, or 1 μL of 400× Gelgreen colorimetric solution is added to the amplification product and placed under an ultraviolet flashlight to analyze the color change in the reaction tube to detect whether the sample contains duck circovirus or duck adenovirus type 3. The detection method can be used to detect whether poultry products are safe, such as for inspection and quarantine of imported poultry.

[0020] In some embodiments of the third aspect of the present invention, the conditions of the loop-mediated isothermal amplification reaction are 65° C. and 60 minutes.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an efficient, accurate and easy-to-operate dual LAMP detection system for duck circovirus and duck adenovirus type 3 and a corresponding kit. The kit does not require complex equipment or professional personnel to operate, can detect duck circovirus and duck adenovirus type 3 at the same time, and can introduce visual detection technology, is suitable for grassroots veterinary laboratories and on-site rapid detection, and provides a new duck circovirus and duck adenovirus type 3 detection method for grassroots veterinarians and farmers to meet the needs of rapid diagnosis of these two viruses in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the electrophoresis diagram during primer screening in Example 2; Figure 2 is the electrophoresis diagram of the optimized dual LAMP detection system in Example 3; Figure 3 is a photograph of the detection result of establishing a visualization kit in Example 4; Figure 4 is a photo of the sensitivity experiment of the kit described in Example 5; Figure 5 This is a photo of the actual sample test results of the visualization kit in Example 6; Figure 6 It is a photo of the specific experiment of the kit described in Example 7. DETAILED DESCRIPTION

[0023] The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the Molecular Cloning Experiment Guide (3rd edition) or in accordance with the methods and product instructions; the biological materials described in the methods, unless otherwise specified, can be obtained from commercial channels.

[0024] Example 1: Primer design for dual LAMP detection system.

[0025] Based on the conserved sequences in the cap gene of duck circovirus and the fiber 2 gene of duck adenovirus type 3, three sets of primers (groups A, B, and C) were designed for duck circovirus and duck adenovirus type 3, respectively. One set of primers included outer primers F3 and B3 and inner primers FIP and BIP. The designed loop-mediated isothermal amplification primer sequences are shown in Table 1.

[0026] Table 1. Primer sequences for dual LAMP reaction

[0027] Example 2: Establishment of dual LAMP detection system and primer screening.

[0028] Since the LAMP method requires too many primers and there may be mutual interference and other unknown factors between primers, detection is required before the experiment to avoid interference from primer interaction. Artificially synthesized DuCV and DAdV-3 plasmids are used as DNA templates, double distilled water is used as blank control, and three sets of designed and synthesized primers are used for single and double LAMP reactions respectively. The labeled and treated samples and negative controls are added to the reagent reaction tubes that have been packaged, the tube caps are tightly closed, the 8-tube strips are vortexed for 3 seconds to mix, and the 8-tube strips are transferred to the detection area after instantaneous centrifugation for 5 seconds.

[0029] The LAMP detection system was 25 μL in total, including 12.5 μL (1×) of 2× LAMP Master Mix, 0.25 μL (20 μmol / L) of outer primers F3 and B3, 1 μL (20 μmol / L) of inner primers FIP and BIP, and 1 μL of template plasmid. ddH2O was added to make up the system and mixed. The initial reaction system was kept at 65°C for 60 minutes. After the amplification, 5 μL of LAMP reaction product was taken for 1.5% agarose gel electrophoresis. The specificity of the primers and the amplification efficiency were analyzed according to the electrophoresis bands.

[0030] The results are as follows Figure 1 shown. Figure 1 The middle (A) shows the detection results of duck circovirus. Lane 1 is the amplification product of primers SEQ ID No: 1~4, Lane 2 is the amplification product of primers SEQ ID No: 5~8, and Lane 3 is the amplification product of primers SEQ ID No: 9~12. The amplification products of Lanes 1 and 2 are ladder-like bands, while the product of Lane 3 has no obvious ladder-like bands, so the primers corresponding to Lanes 1 and 2 are selected as candidate primers.

[0031] Figure 1The middle (B) shows the detection results of duck adenovirus type 3. Lane 1 is the amplification product of primers SEQ ID No: 13~16, Lane 2 is the amplification product of primers SEQ ID No: 17~20, and Lane 3 is the amplification product of primers SEQ ID No: 21~24. Lanes 1 and 2 show ladder-like bands, while the ladder-like bands of the product in Lane 3 are blurred, so the primers corresponding to Lanes 1 and 2 are selected as candidate primers.

[0032] Two sets of candidate primers for duck circovirus and two sets of candidate primers for duck adenovirus type 3 were combined in pairs for double LAMP reaction. The results are as follows: Figure 1 As shown in (C). Lane 1 is the positive amplification product of the dual LAMP mixed primers of SEQ ID No: 1~4 and SEQ ID No: 13~16, Lane 3 is the positive amplification product of the dual LAMP mixed primers of SEQ ID No: 1~4 and SEQ ID No: 17~20, Lane 5 is the positive amplification product of the dual LAMP mixed primers of SEQ ID No: 5~8 and SEQ ID No: 13~16, and Lane 7 is the positive amplification product of the dual LAMP mixed primers of SEQ ID No: 5~8 and SEQ ID No: 17~20; while Lanes 2, 4, 6, and 8 are the negative amplification products of the above corresponding combinations. The ladder-like bands in Lane 1 are fuzzy; Lanes 3 and 4 are ladder-like bands, and it is impossible to distinguish between positive and negative; Lane 5 has a more obvious ladder-like band of dual LAMP mixed primers than Lane 7. Therefore, the double mixed primers composed of SEQ ID No: 5-8 and SEQ ID No: 13-16 for amplifying duck circovirus and duck adenovirus type 3 have better specific amplification effect.

[0033] Example 3: Optimization of the DuCV and DAdV-3 dual LAMP detection system.

[0034] According to the reaction conditions and primer annealing temperature of the LAMP method, the mixed plasmid of duck circovirus and duck adenovirus type 3 was used as a template, and the reaction temperature was set at 60°C, 63°C, 65°C and 68°C for 60 minutes, and the optimal reaction temperature was determined according to the results of agarose gel electrophoresis. Based on the optimized reaction conditions, the reaction was carried out at a reaction time of 60 minutes, 65 minutes, 70 minutes and 75 minutes, and the optimal reaction time was determined according to the results of agarose gel electrophoresis.

[0035] Filter results by Figure 2 As shown in (A), within the reaction temperature range of 60℃~68℃, the bands in lanes 3 and 4 are relatively complete, while the bands in lanes 1 and 2 are relatively fuzzy, so 65℃ was selected as the reaction temperature of the dual LAMP detection system.

[0036] Under the premise of keeping the other reaction conditions unchanged and the reaction temperature at 65°C, the reaction time was changed to 60 minutes to 75 minutes. Figure 2 As shown in the middle (B), the electrophoretic bands did not change much and were relatively similar. Taking into account that Lane 1 took a shorter time, the optimal reaction time for the dual LAMP detection system was 60 minutes.

[0037] Example 4: Establishment of DuCV and DAdV-3 visualization detection kit.

[0038] Based on the optimized dual LAMP detection system, a visualization detection kit was constructed. On the basis of the dual LAMP detection system of Example 3, ultrapure water was used as the negative control for amplification. After the amplification, 1 μL of 400× Gelgreen colorimetric solution was added to the reaction tube cover, and the reaction tube was turned upside down for 5 seconds and oscillated to mix the reaction solution and the colorimetric solution. The result was interpreted based on the color change of the Gelgreen colorimetric solution under an ultraviolet flashlight. On the premise that the negative control reaction tube remains orange-yellow, if the reaction tube of the sample to be tested shows obvious fluorescent bright green, it indicates that the test sample contains duck circovirus or duck adenovirus type 3; if the reaction tube of the sample to be tested is orange-yellow and does not emit fluorescence, it indicates that the test sample does not contain duck circovirus and duck adenovirus type 3. Figure 3 As shown in (A), tube 1 is the double LAMP test result of duck circovirus and duck adenovirus type 3, tube 2 is the LAMP test result of duck circovirus alone, and tube 3 is the LAMP test result of duck adenovirus type 3. All of the above reaction tubes show obvious bright green fluorescence, while tube 4 is the negative control, which is orange and does not emit fluorescence. The above results are consistent with Figure 3 The agarose gel electrophoresis results in (B) are consistent. This proves that the addition of Gelgreen color developing solution can make the kit visualized, which helps users to perform qualitative analysis of samples in the absence of other equipment.

[0039] Example 5: Sensitivity test of the kit.

[0040] The synthesized positive control quality control product (containing DuCV plasmid and DAdV-3 plasmid) was diluted 10 times in sequence as a DNA template, with concentrations of 0.8 ng, 0.08 ng, 0.008 ng and 0.0008 ng, respectively, and amplified according to the dual LAMP detection system optimized in Example 3. After the amplification, 1 μL of 400× Gelgreen dye was added to the amplified products, and the sensitivity of the method was determined by the color change of the dye under an ultraviolet flashlight. The results are shown in Figure 4As shown, tubes 1 to 3 can all be observed to have obvious bright green fluorescence, and tube 4 is orange-yellow and does not emit any fluorescence. Therefore, the sensitivity of the duck circovirus and duck adenovirus type 3 dual detection kit is 0.008 ng.

[0041] Example 6: Kit detection example.

[0042] In order to verify the accuracy and practicality of the dual detection kit for duck circovirus and duck adenovirus type 3, suspected infected samples were collected from duck farms and pretreated, and nucleic acids were extracted using a viral DNA kit as reaction templates, and amplified according to the optimized dual LAMP detection system. Positive and negative samples were used as controls, and the color change of Gelgreen dye and the agarose gel electrophoresis bands were observed to determine whether the samples contained duck circovirus or duck adenovirus type 3. The results are shown in Figure 2. Figure 5 As shown, Figure 5 In the middle (A), tubes 1 and 2 showed obvious fluorescent bright green, indicating that the sample contained duck circovirus or duck adenovirus type 3, while tube 3 showed orange-yellow and did not emit fluorescent color, indicating negative; tubes 4 and 5 were positive and negative controls, respectively. Figure 5 The agarose gel electrophoresis results in the middle (B) are consistent, indicating that the results of the visualization detection method are reliable and the sample can be qualitatively judged directly by the color of the amplification product, greatly shortening the detection time.

[0043] Example 7: Specificity test of the kit.

[0044] The DNA extracted from duck circovirus, duck adenovirus type 3, duck parvovirus and duck plague virus was used as templates, and the duck circovirus and duck adenovirus type 3 dual detection kit was used for amplification. The specificity of the method was verified by adding Gelgreen dye to the amplified product and observing the color change. The test results are shown in Figure 6 As shown, tubes 1 to 3 using duck circovirus or duck adenovirus type 3 as templates all showed obvious bright green fluorescence, while duck parvovirus in tube 4, duck plague virus in tube 5, and negative control in tube 6 did not show obvious bright green fluorescence, indicating that this method has strong specificity for duck circovirus and duck adenovirus type 3 and is not easily interfered by other pathogens.

[0045] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A dual LAMP detection system, characterized in that: The invention comprises a primer set, which comprises primers for specifically detecting duck circovirus cap gene: DuCV-F3, DuCV-B3, DuCV-FIP and DuCV-BIP; and primers for specifically detecting duck adenovirus type 3 fiber 2 gene: DAdV-3-F3, DAdV-3-B3, DAdV-3-FIP and DAdV-3-BIP.

2. The dual LAMP detection system according to claim 1, characterized in that: The nucleotide sequence of the DuCV-F3 is shown in SEQ ID No:5; the nucleotide sequence of the DuCV-B3 is shown in SEQ ID No:6; the nucleotide sequence of the DuCV-FIP is shown in SEQ ID No:7; the nucleotide sequence of the DuCV-BIP is shown in SEQ ID No:8; the nucleotide sequence of the DAdV-3-F3 is shown in SEQ ID No:13; the nucleotide sequence of the DAdV-3-B3 is shown in SEQ ID No:14; the nucleotide sequence of the DAdV-3-FIP is shown in SEQ ID No:15; the nucleotide sequence of the DAdV-3-BIP is shown in SEQ ID No:

16.

3. The dual LAMP detection system according to claim 2, characterized in that: The molar ratio of the DuCV-F3, the DuCV-B3, the DuCV-FIP and the DuCV-BIP is 1:1:4:

4.

4. The dual LAMP detection system according to claim 2, characterized in that: The molar ratio of the DAdV-3-F3, the DAdV-3-B3, the DAdV-3-FIP and the DAdV-3-BIP is 1:1:4:

4.

5. The dual LAMP detection system according to claim 1, characterized in that: A loop-mediated isothermal amplification buffer is also included.

6. A dual detection kit for duck circovirus and duck adenovirus type 3, characterized in that: It comprises the dual LAMP detection system as claimed in any one of claims 1 to 5.

7. The kit according to claim 6, characterized in that It also includes at least one of Gelgreen color developing solution, positive control quality control product and negative control quality control product.

8. The kit according to claim 7, characterized in that: The positive control quality control product is a mixed solution of a plasmid containing the duck circovirus cap gene and a plasmid containing the duck adenovirus type 3 Fiber2 gene.

9. A detection method for a non-disease diagnosis purpose of the kit according to any one of claims 6 to 8, characterized in that: Includes steps: (1) Extract DNA from the sample to be tested as a reaction template; (2) Mixing the primer set, the loop-mediated isothermal amplification buffer and the reaction template to perform a loop-mediated isothermal amplification reaction.

10. The detection method according to claim 9, characterized in that: The conditions of the loop-mediated isothermal amplification reaction were 65° C. for 60 minutes.

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