A LAMP primer set and kit for detecting human circovirus

Abstract

The present invention discloses a LAMP primer set and a kit for detecting human circovirus. The present invention is directed to human circovirus type 1 and type 2, and designs a LAMP primer set that can specifically detect human circovirus, including a LAMP primer set for detecting human circovirus type 1 and / or a LAMP primer set for detecting human circovirus type 2. The LAMP primer set of the present invention has the advantages of low detection limit, high specificity and good sensitivity, and can realize rapid and accurate screening of human circovirus. On this basis, the present invention also provides a kit containing the above-mentioned LAMP primer set for detecting human circovirus, which can realize the detection of human circovirus in a short time, and the operation is simple, and the result is easy to determine, which is conducive to the rapid detection of human circovirus.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and more specifically, relates to a LAMP primer set and a kit for detecting human circovirus. Background Art

[0002] Circoviruses are a class of non-enveloped, single-stranded DNA viruses with circular symmetry that have been found in a variety of hosts, including birds, fish, insects, and mammals. Porcine circovirus (PCV) is the smallest known virus in the Circoviridae family. Although direct evidence of zoonotic transmission of PCV from animals to humans is lacking, some studies have reported the detection of PCV1 or PCV2 in human samples. In addition, PCV3 has been shown to be able to infect non-human primates. These findings indicate the potential risk of zoonotic transmission of circoviruses and that new circoviruses may exist in humans. Indeed, two new circoviruses that can infect humans have been discovered.

[0003] The Pasteur Institute in France reported a new type of circovirus that can infect humans, named human circovirus type 1 (HCirV-1, hereinafter referred to as HuCV-1). Other researchers detected viral sequences related to PCV3 and assembled a complete circular genome. Based on phylogenetic analysis and genetic distance analysis, it was proved that the virus may represent a new type of human circovirus (HuCV-2).

[0004] Human circovirus (HuCV) is a new virus that is pathogenic to humans. The correlation between the virus and hepatitis cannot be ruled out, but its mode of transmission is still unclear. When the infection is still unexplained, it is crucial to identify a new pathogen and develop a corresponding detection method. That is, the development of early rapid diagnostic reagents for HuCV virus infection plays a key role in the prevention and treatment of some diseases caused by the infection. Although there are fluorescent quantitative qPCR detection primers and methods for human circovirus type 1 and type 2 in the prior art, the detection based on fluorescent quantitative PCR requires expensive instruments, and the PCR detection time is long, which is not conducive to promotion and application.

[0005] Loop-mediated isothermal amplification (LAMP) has the characteristics of simple operation, easy product detection, and low requirements for instruments and equipment. The reaction can be achieved using a water bath or a constant temperature box, and the results of the loop-mediated isothermal amplification reaction can be judged by the naked eye, which is suitable for rapid diagnosis at the grassroots level. Since the genome sequence of human circovirus is short, only 2000nt, it is very difficult to design LAMP primers that can simultaneously distinguish HuCV-1 and HuCV-2 in a limited segment. At present, LAMP primer sets and corresponding detection kits that can quickly and accurately detect human circovirus have not been developed. Summary of the invention

[0006] Aiming at the deficiencies of the existing human circovirus detection technology, the present invention provides a LAMP primer set and a kit for detecting human circovirus.

[0007] The first object of the present invention is to provide a LAMP primer set for detecting human circovirus type 1.

[0008] The second object of the present invention is to provide a LAMP primer set for detecting human circovirus type 2.

[0009] The third object of the present invention is to provide a LAMP primer set for detecting human circovirus.

[0010] The fourth object of the present invention is to provide the use of the above-mentioned LAMP primer set for detecting human circovirus in the preparation of a human circovirus detection reagent or kit.

[0011] The fifth object of the present invention is to provide a LAMP kit for detecting human circovirus. The above object of the present invention is achieved by the following technical solutions:

[0012] The present invention designs LAMP primer sets for specific detection of HuCV-1 or HuCV-2 respectively for the CAP gene sequences of human circovirus type 1 (HuCV-1, genome accession number GenBank: ON677309) and human circovirus type 2 (HuCV-2, genome accession number GenBank: ON226770). In addition, the LAMP primer sets can also be used to simultaneously detect HuCV-1 and HuCV-2 in a single-tube reaction.

[0013] The present invention provides a LAMP primer set for detecting human circovirus type 1, comprising an outer primer F3 shown in SEQ ID NO:1, an outer primer B3 shown in SEQ ID NO:2, an inner primer FIP shown in SEQ ID NO:3, an inner primer BIP shown in SEQ ID NO:4, a loop primer LF shown in SEQ ID NO:5 and a loop primer LB shown in SEQ ID NO:6.

[0014] The present invention also provides a LAMP primer set for detecting human circovirus type 2, comprising an outer primer F3 shown in SEQ ID NO:7, an outer primer B3 shown in SEQ ID NO:8, an inner primer FIP shown in SEQ ID NO:9, an inner primer BIP shown in SEQ ID NO:10 and a loop primer LB-3 shown in SEQ ID NO:11.

[0015] The present invention also provides a LAMP primer set for detecting human circovirus, comprising the above-mentioned LAMP primer set for detecting human circovirus type 1 and type 2.

[0016] Specifically, the loop primer LF shown in SEQ ID NO: 5 and / or the loop primer LB-3 shown in SEQ ID NO: 11 has a quenching group at its 5' end and a fluorescent group at its 3' end.

[0017] Optionally, the fluorescent group is AMCA, Pacific Blue, Atto 425, BODIPY FL, FAM, Alexa Fluor488, TET, JOE, Yakima Yellow, VIC, HEX, Quasar 570, Cy3, NED, TAMRA, ROX, AquaPhluor593, Texas Red, Atto 590, Cy5, Quasar 670 or Cy5.5.

[0018] Optionally, the quencher group is BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse or MGB.

[0019] Specifically, the loop primer LF shown in SEQ ID NO: 5 is connected to BHQ2 at its 5' end and to Cy5 at its 3' end.

[0020] Specifically, the loop primer LB-3 shown in SEQ ID NO: 11 has BHQ2 at its 5' end and VIC at its 3' end.

[0021] Since the LAMP primer set of the present invention can be used to specifically detect human circovirus, the present invention also seeks to protect the use of the LAMP primer set in preparing a reagent or kit for detecting human circovirus.

[0022] The present invention also provides a LAMP kit for detecting human circovirus, wherein the kit contains primers shown in SEQ ID NOs: 1 to 11.

[0023] Specifically, the kit also contains: sample lysis solution, positive control, negative control and reagents required for isothermal amplification reaction.

[0024] Optionally, the negative control is sterile water.

[0025] Optionally, the positive control is a recombinant plasmid containing the HuCV-1CAP gene sequence, named puc-HuCV-1; and a recombinant plasmid containing the HuCV-2CAP gene sequence, named puc-HuCV-2.

[0026] Specifically, the reagents required for the isothermal amplification reaction include: Neb Q5 high-fidelity enzyme, DNA polymerase with strand displacement ability, dNTP, MgSO4 and 10×buffer buffer.

[0027] Specifically, the 10× isothermal amplification buffer contains the following components at the following concentrations: 100 mM Tris-HCl (pH 8.5), 500 nM KCl, and 15 nM MgCl2.

[0028] Specifically, the DNA polymerase is Bst DNA polymerase.

[0029] Specifically, the kit also contains a color developing solution.

[0030] Optionally, the color developing solution is phenol red, neutral red or cresyl violet.

[0031] The present invention also provides a method for quickly identifying the LAMP result, wherein a color developing solution is added to the LAMP reaction product. If the color developing solution shows that the LAMP reaction product is alkaline, the test result is positive. The principle is that during the DNA synthesis process of the LAMP reaction, the byproducts released include pyrophosphate and H + Therefore, the pH value of the reaction solution will change as the reaction proceeds, from the initial alkalinity to the acidity. By using pH-sensitive indicators as dyes to detect DNA amplification, the amplification results can be visually judged by the naked eye to distinguish between positive and negative results.

[0032] Specifically, the LAMP reaction system prepared using the kit of the present invention is: taking 25 μL isothermal amplification system as an example, 2.5 μL of 10× isothermal amplification system buffer, 1 μL of 8U / μL Bst DNA polymerase, 1 μL of 100 mM MgS04, 3.5 μL of 10 mM dNTP, 0.5 μL of 10 μM primer F3, 0.5 μL of 10 μM primer B3, 0.5 μL of 80 μM primer FIP, 0.5 μL of 80 μM primer BIP0.5 μL, 0.5 μL of 40 μM primer LF, 0.5 μL of 40 μM LB primer, 3 μL of template, and supplemented to 25 μL with nuclease-free water.

[0033] If the primer set does not contain primer LF, do not add this component when preparing the reaction system, and use nuclease-free water to make up the reaction system.

[0034] Specifically, the LAMP isothermal amplification reaction conditions are: 50-65° C. for 10-60 min.

[0035] Preferably, the optimal reaction conditions are: 65°C for 50 min.

[0036] The present invention has the following beneficial effects:

[0037] The present invention provides a LAMP primer set and a kit for detecting human circovirus. By using the LAMP primer set of the present invention, not only can the typing and co-detection of human circovirus type 1 and type 2 be achieved, but also only constant temperature amplification is required during the detection process, and no expensive amplification instruments and complicated amplification procedures are required like PCR, and the detection can be completed within 50 minutes, which has the advantages of high efficiency and rapidity. At the same time, the specificity and sensitivity of the human circovirus detected by the LAMP primer set of the present invention are good, and the detection limit is as low as 1 copies / μL, and the concentration of 10 copies / μL or more can be stably detected, so that the human circovirus is sensitive, specific and accurate. In addition, the present invention also provides a kit for detecting human circovirus, which can realize the detection of human circovirus in a short time by using the kit, and the operation is simple, and the result is easy to determine, which is conducive to the rapid detection of human circovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the sensitivity test result of isothermal amplification of HuCV-2CAP gene by CAP2-4-1 primer set.

[0039] Figure 2 The sensitivity test results of the five LAMP primer sets designed for the HuCV-1CAP gene in Table 1.

[0040] Figure 3The sensitivity test results of the 9 LAMP primer sets designed for the HuCV-2CAP gene in Table 2.

[0041] Figure 4 This is the sensitivity test result of isothermal amplification of HuCV-1CAP gene by CAP1-2 primer set.

[0042] Figure 5 This is the sensitivity test result of isothermal amplification of HuCV-2CAP gene by CAP2-4-3 primer set.

[0043] Figure 6 This is the sensitivity test result of isothermal amplification of HuCV-1CAP gene using CAP1-2 primer set combined with CAP2-4-3 primer set.

[0044] Figure 7 This is the sensitivity test result of isothermal amplification of HuCV-2CAP gene using CAP1-2 primer set combined with CAP2-4-3 primer set.

[0045] Figure 8 The results are shown in Figure 2, which are the results of amplification of the CAP genes of HuCV-1 and HuCV-2 using the CAP-1-2 primer set.

[0046] Fig. 9 The results are shown in Figure 2, which are the results of amplification of the CAP genes of HuCV-1 and HuCV-2 using the CAP-2-4-3 primer set.

[0047] Fig.10 The results show the specificity of isothermal amplification of 8 common viruses by using the CAP1-2 and CAP2-4-3 primer sets. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0049] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0050] Example 1 LAMP primer design

[0051] The present invention designs multiple groups of specific LAMP primer sets according to the two types of CAP genes of HuCV-1 (accession number GenBank: ON677309) and HuCV-2 (accession number GenBank: ON226770) reported in NCBI, and selects several groups with better preliminary experimental effects; wherein, the primer set for detecting HuCV-1 includes: CAP-1-1, CAP-1-2, CAP-1-3, CAP-1-4 and CAP-1-5; the primer set for detecting HuCV-2 includes: CAP-2-1, CAP-2-2, CAP-2-3, CAP-2-4-1 and CAP-2-5. It is found in the preliminary experiment that the amplification effect of the CAP-2-4-1 primer set is better, but the specificity is not enough. Therefore, the present invention optimizes the LB primer sequence in the CAP-2-4-1 primer set, and designs the CAP-2-4-2, CAP2-4-3, CAP2-4-4 and CAP2-4-5 primer sets, wherein the F3, B3, FIP and BIP primers are consistent with the primer sequences in the CAP-2-4-1 primer set, and only the LB sequence is different. The specific sequences (5'-3') of the above primers are shown in Tables 1 and 2 below, respectively:

[0052] Table 1 LAMP primer sets for detecting HuCV-1 virus

[0053]

[0054]

[0055] Table 2 LAMP primer sets for detecting HuCV-2 virus

[0056]

[0057]

[0058] When the above LAMP primer set is used for detection, the 5' end of one of the loop primers needs to be connected to a quenching group, and the 3' end needs to be connected to a fluorescent group.

[0059] Optionally, the fluorescent group is AMCA, Pacific Blue, Atto 425, BODIPY FL, FAM, Alexa Fluor 488, TET, JOE, Yakima Yellow, VIC, HEX, Quasar 570, Cy3, NED, TAMRA, ROX, AquaPhluor 593, Texas Red, Atto 590, Cy5, Quasar 670 or Cy5.5. The quenching group is BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse or MGB.

[0060] Example 2 Screening of LAMP primer sets

[0061] The concentrations of plasmids puc-HuCV-1 and puc-HuCV-2 (synthesized by General Biotech) containing the target gene (CAP gene) were measured using NanoDrop One / OneC micro-nucleic acid protein concentration meter. puc-HuCV-2 was diluted 10 times in a gradient to 1000 copies / μL, 100 copies / μL, 10 copies / μL, and 1 copy / μL. The samples of all dilution multiples were used as templates, and the LAMP isothermal amplification reaction was performed using the CAP2-4-1 primer set. Each reaction was repeated 2 times. The LAMP isothermal amplification reaction system is shown in Table 3. The LAMP isothermal amplification reaction conditions are: 10 to 60 minutes at 50 to 65°C. After testing, the optimal reaction conditions are: 65°C for 50 minutes, and this reaction condition is used in this embodiment.

[0062] Table 3 LAMP single primer set isothermal amplification reaction system

[0063] Serial number Components Concentration / μL 1 10× Constant Temperature Amplification Buffer 2.5 2 <![CDATA[100mM MgSO4]]> 2 3 10mM dNTP 3.5 4 10 μM Primer F3 0.5 5 10 μM Primer B3 0.5 6 80 μM Primer FIP 0.5 7 80 μM Primer BiP 0.5 8 20 μM Primer LB 0.5 9 20 μM Primer LF 0.5 10 8U Bst polymerase 1 11 NEB Q5 High Fidelity Enzyme 0.075 12 template 3 13 Nuclease-free water Up to 25 μL

[0064] Not all primer sets have sequence number 8 (LB primer) and sequence number 9 (LF primer). For reaction systems that do not have sequence number 8 (LB primer) and sequence number 9 (LF primer) components, use nuclease-free water to replace them, that is, use nuclease-free water to make up to 25 μL of the reaction system.

[0065] Specifically, the 10× isothermal amplification buffer contains the following components at the following concentrations: 100 mM Tris-HCl (pH 8.5), 500 nM KCl, and 15 nM MgCl2.

[0066] The sensitivity test results of isothermal amplification of HuCV-2CAP gene by CAP2-4-1 primer set are as follows Figure 1 As shown. Figure 1 It can be seen that although the CAP-2-4-1 primer set is effective for 1 copies / μL (10 0 The templates with a concentration of 100 copies / μL showed amplification signals, which indicated a high sensitivity. However, the amplification curve of the negative control (NTC) also showed signals, indicating that the specificity of the primer set corresponding to CAP-2-4-1 was not high. Therefore, the LB primer sequence of CAP-2-4-1 was optimized, and the LB-2, LB-3, LB-4, and LB5 loop primers were designed, which were combined with the F3 primer, B3 primer, FIP primer, and BIP primer of CAP-2-4-1 to form the CAP-2-4-2, CAP-2-4-3, CAP-2-4-4, and CAP-2-4-5 primer sets.

[0067] 5 groups of LAMP primer sets for detecting HuCV-1 and 9 groups of LAMP primer sets for detecting HuCV-2 (a total of 14 groups, as shown in Table 1 and Table 2, respectively) were optimized. Among them, the puc-HuCV-1 plasmid was used as the amplification template for the HuCV-1 virus detection primer set, and the puc-HuCV-2 plasmid was used as the amplification template for the HuCV-2 virus detection primer set, and the template concentration was uniformly 1000 copies / μL. The LAMP isothermal amplification reaction system is shown in Table 3, and the LAMP isothermal amplification reaction conditions are 65°C for 50 minutes. The amplification efficiency of each primer set was compared under the same conditions.

[0068] The sensitivity test results of the five LAMP primer sets designed for the HuCV-1CAP gene in Table 1 are as follows Figure 2 As shown. Figure 2 It can be seen that at the same template concentration, the CAP-1-2 primer set has the shortest amplification curve time compared with other primer sets. The sensitivity test results of the 9 sets of LAMP primer sets designed for the HuCV-2CAP gene in Table 2 are as follows: Figure 3 As shown, from Figure 3 It can be seen that after changing the loop primer sequence, the CAP-2-4-3 primer set did not show NTC jump, indicating that the CAP-2-4-3 primer set has strong specificity and amplification signal appears quickly, so the CAP-2-4-3 primer set was finally selected. CAP-1-2 and CAP-2-4-3 were selected as the preferred primer sets, and these two primer sets were used for subsequent experiments.

[0069] The present invention also provides a kit for detecting human circovirus, wherein the kit contains primer set CAP-1-2 and / or CAP-2-4-3, wherein the nucleotide sequences of the primers contained in the primer set CAP-1-2 are shown in SEQ ID NOs: 1 to 6; and the nucleotide sequences of the primers contained in the primer set CAP-2-4-3 are shown in SEQ ID NOs: 7 to 11. In addition to the above primer set, the kit also contains 10× isothermal amplification system buffer, 8U / μL Bst DNA polymerase, 100mM MgS04, 10mM dNTP, sample lysate, positive control (puc-HuCV-1 and puc-HuCV-2 plasmids), and negative control (sterile water or nuclease-free water).

[0070] Example 3 Sensitivity detection of single-tube single-target CAP1-2 primer set and CAP2-4-3 primer set

[0071] After optimizing the primers, the concentrations of the puc-HuCV-1 plasmid and the puc-HuCV-2 plasmid were measured using a NanoDrop One / OneC micro-nucleic acid protein concentration meter, and then the samples were diluted in a 10-fold gradient to 1000 copies / μL, 100 copies / μL, 10 copies / μL, and 1 copy / μL. The samples of all dilution multiples were used as templates and amplified using the LAMP detection kit provided by the present invention, with 2 replicates for each reaction.

[0072] The LAMP amplification system refers to the LAMP isothermal amplification reaction system in Table 3 of Example 2.

[0073] The reaction conditions are: 65°C for 50 min, single-tube single-target reaction, that is, the single-tube reaction system contains only puc-HuCV-1 plasmid or puc-HuCV-2 plasmid and the corresponding CAP1-2 primer set or CAP2-4-3 primer set.

[0074] The sensitivity test results of isothermal amplification of HuCV-1CAP gene by CAP1-2 primer set and the sensitivity test results of isothermal amplification of HuCV-2CAP gene by CAP2-4-3 primer set are shown as follows: Figure 4 and Figure 5 As shown. Figure 4 and 5 It can be seen that the negative control test results are normal, with no fluorescent signal, while the positive sample has a fluorescent signal, indicating that the primer set of the present invention can detect HuCV-1 or HuCV-2. Combined with the results of two repeated tests, it can be seen that the primer set of the present invention can detect 1 copies / μL probability and all other concentrations of DNA templates. According to the conversion of 3μL sample volume, concentrations above 10 copies / reaction can be stably detected.

[0075] Example 4 Sensitivity Detection of Single-Tube Dual-Target CAP-1-2 Primer Set and CAP-2-4-3 Primer Set

[0076] The puc-HuCV-1 plasmid and puc-HuCV-2 plasmid diluted 10 times to 1000 copies / μL, 100 copies / μL, 10 copies / μL, and 1 copy / μL were used as templates, and the CAP-1-2 and CAP-2-4-3 primer sets were placed in the same reaction tube for isothermal amplification to observe whether the combined use of the two primer sets would affect the detection sensitivity of human circovirus type 1 or type 2. Two replicates were set for each reaction. The LAMP isothermal amplification reaction system is shown in Table 4.

[0077] Table 4 LAMP dual primer set isothermal amplification reaction system

[0078]

[0079]

[0080] The sensitivity test results of isothermal amplification of HuCV-1CAP gene by CAP1-2 primer set combined with CAP2-4-3 primer set are as follows Figure 6 As shown, the sensitivity test results of isothermal amplification of HuCV-2CAP gene by CAP1-2 primer set combined with CAP2-4-3 primer set are as follows Figure 7 As shown. Figure 6 and Figure 7 It can be seen that the CAP-1-2 primer set can detect a target concentration of 1 copies / μL, and the CAP-2-4 primer set can detect a target concentration of 1 copies / μL, indicating that the combined use of the CAP-1-2 primer set and the CAP-2-4-3 primer set will not affect their sensitivity, and the CAP-1-2 and CAP-2-4-3 primer sets can jointly detect human circovirus type 1 and type 2.

[0081] Example 5 Specific detection of single-tube single-target CAP-1-2 primer set and CAP-2-4-3 primer set

[0082] In order to detect the detection specificity of the CAP-1-2 primer set and the CAP-2-4-3 primer set, the CAP-1-2 primer set was used to amplify the puc-HuCV-1 plasmid and the puc-HuCV-2 plasmid, respectively, and the CAP-1-2 primer set was also used to amplify the puc-HuCV-1 plasmid and the puc-HuCV-2 plasmid, respectively. The amplification system and conditions were the same as in Example 2.

[0083] The results of the amplification reaction of the HuCV-1CAP gene and the HuCV-2CAP gene by the CAP-1-2 primer set are shown in Figure 8 As shown in the figure, the results of the amplification reaction of the HuCV-1CAP gene and the HuCV-2CAP gene by the CAP-2-4-3 primer set are as follows Fig. 9 As shown. Figure 8 and 9 It can be seen that the CAP1-2 primer group only has an amplification signal for the puc-HuCV-1 plasmid template, and the CAP2-4-3 primer group only has an amplification signal for the puc-HuCV-2 plasmid template. No amplification signal was detected after cross-detection, indicating that the CAP-1-2 and CAP-2-4-3 primer groups have high specificity and can realize the typing detection of the two human circoviruses.

[0084] Example 6 Specific detection of different viruses by single-tube dual-target CAP-1-2 and CAP-2-4-3 primer sets

[0085] In order to detect the detection specificity of the CAP-1-2 primer set and the CAP-2-4-3 primer set for other common human viruses, this example used DNA of 8 common human viruses, puc-HuCV-1 plasmid and puc-HuCV-2 plasmid as templates for detection; wherein, the addition amount of each template was 1 μL, the CAP-1-2 and CAP-2-4-3 primer sets were placed in the same reaction tube, and the amplification system and conditions were the same as in Example 4. The 8 common human viruses are: BK polyomavirus, JC polyomavirus, cytomegalovirus, human adenovirus type 5, human immunodeficiency virus (HIV1), hepatitis B virus, hepatitis C virus and hepatitis G virus.

[0086] The specificity test results of CAP1-2 and CAP2-4-3 primer sets for amplifying 8 common viruses are shown in the figure below. Fig.10 As shown. Fig.10 It can be seen that, except for HuCV-1 and HuCV-2, no amplification signals were detected for other viral DNA, indicating that the specificity of the CAP-1-2 and CAP-2-4-3 primer sets is high and can specifically detect human circovirus. In addition, the present invention also compares the CAP1-2 and CAP2-4-3 primer sets with the CAP gene in PCV, and the results show that the interval anchored by the CAP1-2 and CAP2-4-3 primer sets has a huge difference in the matching rate with the CAP gene in PCV, that is, the CAP1-2 and CAP2-4-3 primer sets described in the present invention will not amplify PCV, and have high specificity for human circovirus.

[0087] In summary, the LAMP detection primer set, kit and detection method for HuCV-1 and HuCV-2 human circovirus provided in the present application can complete the detection of new human circovirus within 50 minutes. It has a fast detection speed and is easy to operate, and can be widely used in the rapid differential diagnosis of new human circovirus.

[0088] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A LAMP primer set for detecting human circovirus type 1, characterized in that: It includes an outer primer F3 shown in SEQ ID NO:1, an outer primer B3 shown in SEQ ID NO:2, an inner primer FIP shown in SEQ ID NO:3, an inner primer BIP shown in SEQ ID NO:4, a loop primer LF shown in SEQ ID NO:5 and a loop primer LB shown in SEQ ID NO:

6.

2. A LAMP primer set for detecting human circovirus type 2, characterized in that: It includes an outer primer F3 shown in SEQ ID NO:7, an outer primer B3 shown in SEQ ID NO:8, an inner primer FIP shown in SEQ ID NO:9, an inner primer BIP shown in SEQ ID NO:10 and a loop primer LB-3 shown in SEQ ID NO:

11.

3. A LAMP primer set for detecting human circovirus type 1 and / or human circovirus type 2, characterized in that: The invention comprises the LAMP primer set according to claims 1 and 2.

4. The LAMP primer set according to claim 1, characterized in that The loop primer LF shown in SEQ ID NO: 5 has a quenching group connected to its 5' end and a fluorescent group connected to its 3' end.

5. The LAMP primer set according to claim 2, characterized in that The loop primer LB-3 shown in SEQ ID NO: 11 has a quenching group at its 5' end and a fluorescent group at its 3' end.

6. The LAMP primer set according to claim 3, characterized in that The loop primer LF shown in SEQ ID NO:5 has a quenching group at its 5' end and a fluorescent group at its 3' end; the loop primer LB-3 shown in SEQ ID NO:11 has a quenching group at its 5' end and a fluorescent group at its 3' end.

7. The LAMP primer set according to claim 4, characterized in that The fluorescent group is AMCA, Pacific Blue, Atto 425, BODIPY FL, FAM, Alexa Fluor 488, TET, JOE, Yakima Yellow, VIC, HEX, Quasar 570, Cy3, NED, TAMRA, ROX, Aqua Phluor593, Texas Red, Atto 590, Cy5, Quasar 670 or Cy5.

5.

8. The LAMP primer set according to claim 5, characterized in that The fluorescent group is AMCA, Pacific Blue, Atto 425, BODIPY FL, FAM, Alexa Fluor 488, TET, JOE, Yakima Yellow, VIC, HEX, Quasar 570, Cy3, NED, TAMRA, ROX, Aqua Phluor593, Texas Red, Atto 590, Cy5, Quasar 670 or Cy5.

5.

9. The LAMP primer set according to claim 6, characterized in that: The fluorescent group is AMCA, Pacific Blue, Atto 425, BODIPY FL, FAM, Alexa Fluor 488, TET, JOE, Yakima Yellow, VIC, HEX, Quasar 570, Cy3, NED, TAMRA, ROX, Aqua Phluor593, Texas Red, Atto 590, Cy5, Quasar 670 or Cy5.

5.

10. The LAMP primer set according to claim 4, characterized in that: The quenching group is BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse or MGB.

11. The LAMP primer set according to claim 5, characterized in that The quenching group is BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse or MGB.

12. The LAMP primer set according to claim 6, characterized in that The quenching group is BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse or MGB.

13. Use of the LAMP primer set according to any one of claims 1, 3, 4, 6, 7, 9, 10, and 12 in preparing a human circovirus type 1 detection reagent or kit.

14. Use of the LAMP primer set according to any one of claims 2, 3, 5, 6, 8, 9, 11, and 12 in preparing a human circovirus type 2 detection reagent or kit.

15. A LAMP kit, characterized in that: Contains the LAMP primer set according to any one of claims 1 to 12.

16. The LAMP kit according to claim 15, characterized in that The following components are also included: sample lysis solution, positive control, negative control, and reagents required for isothermal amplification reaction.

17. The LAMP kit according to claim 16, characterized in that The reagents required for the isothermal amplification reaction include the following components: Neb Q5 high-fidelity enzyme, DNA polymerase, dNTP, MgSO4 and 10×buffer buffer.

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