CN117165723A - LAMP primer group and kit for detecting human circovirus - Google Patents
LAMP primer group and kit for detecting human circovirus Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The application discloses an LAMP primer group and a kit for detecting human circovirus. The application designs and obtains the LAMP primer group capable of specifically detecting the human circovirus aiming at the type 1 and the type 2 of the human circovirus, and the LAMP primer group comprises the LAMP primer group for detecting the type 1 of the human circovirus and/or the LAMP primer group for detecting the type 2 of the human circovirus. The LAMP primer group has the advantages of low detection limit, high specificity and good sensitivity, and can realize rapid and accurate screening of the human circovirus. On the basis, the application also provides a kit containing the LAMP primer group for detecting the human circovirus, and the kit can be used for detecting the human circovirus in a short time, is simple to operate, is easy to judge, and is beneficial to rapid detection of the human circovirus.
Description
Technical Field
The application belongs to the technical field of virus detection. More specifically, it relates to a LAMP primer set and a kit for detecting human circovirus.
Background
Circoviruses are a non-enveloped single-stranded DNA virus with circular symmetry that has been found in a variety of hosts, including birds, fish, insects, mammals, and the like. Porcine Circovirus (PCV) is the smallest virus known from the circoviridae family, and although there is a lack of direct evidence of co-transmission of PCV from animals to humans, some studies report the detection of PCV1 or PCV2 in human samples. Furthermore, PCV3 has been demonstrated to be capable of infecting non-human primates. These findings indicate that there is a potential risk of zoonotic transmission of circoviruses, and that new circoviruses may be present in humans. It has also been found that 2 novel circoviruses infect humans.
The French Pasteur institute reported a novel circovirus which was able to infect humans and was designated human circovirus type 1 (HCirV-1, hereinafter HuCV-1). Other researchers detected viral sequences associated with PCV3 and assembled a complete circular genome. Based on phylogenetic analysis and genetic distance analysis, it was demonstrated that the virus may represent a novel human circovirus (HuCV-2).
Human circovirus (HuCV) is a novel virus pathogenic to humans, and the correlation between the virus and hepatitis has not been excluded, but the transmission mode is not yet clarified. It is important to be able to identify a new pathogen and develop a corresponding detection method when the infection is still unexplained. I.e. the development of early rapid diagnostic reagents for HuCV virus infection has a critical role in the control of some diseases caused by such infection. In the prior art, although the detection primers and the detection method aiming at the fluorescent quantitative qPCR of the type 1 and the type 2 of the human circovirus exist, the detection based on the fluorescent quantitative PCR needs expensive instruments, and the PCR detection time is long, so that the method is not beneficial to popularization and application.
The loop-mediated isothermal amplification (LAMP) technology has the characteristics of simple operation, easy detection of products, low requirements on instruments and equipment and the like, the reaction can be realized by utilizing a water bath or an incubator, the result of the loop-mediated isothermal amplification reaction can be judged by naked eyes, and the method is suitable for rapid diagnosis of a base layer. Because the genome sequence of the human circovirus is short and only 2000nt, it is very difficult to design and obtain LAMP primers capable of distinguishing HuCV-1 and HuCV-2 in a limited section at the same time, and a LAMP primer group and a corresponding detection kit capable of rapidly and accurately detecting the human circovirus are not developed at present.
Disclosure of Invention
Aiming at the defects of the existing technology for detecting the human circovirus, the application provides an LAMP primer group and a kit for detecting the human circovirus.
The first object of the present application is to provide a LAMP primer group for detecting human circovirus type 1.
The second object of the present application is to provide a LAMP primer group for detecting human circovirus type 2.
The third object of the present application is to provide a LAMP primer group for detecting human circovirus.
The fourth object of the application is to provide the application of the LAMP primer group for detecting the human circovirus in preparing a human circovirus detection reagent or a kit.
The fifth object of the application is to provide a LAMP kit for detecting human circovirus. The above object of the present application is achieved by the following technical scheme:
the application designs LAMP primer groups capable of specifically detecting HuCV-1 or HuCV-2 respectively aiming at CAP gene sequences of human circovirus type 1 (HuCV-1, genome accession number GenBank: ON 677309) and human circovirus type 2 (HuCV-2, genome accession number GenBank: ON 226770). In addition, detection of HuCV-1 and HuCV-2 can be achieved simultaneously in a single tube reaction using the LAMP primer group.
The application provides an LAMP primer group for detecting human circovirus type 1, which comprises 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.
The application also provides a LAMP primer group for detecting the human circovirus type 2, which comprises 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.
The application also provides an LAMP primer group for detecting the human circovirus, which comprises the LAMP primer group for detecting the human circovirus type 1 and type 2.
Specifically, the 5' end of the loop primer LF shown in SEQ ID NO. 5 and/or the 5' end of the loop primer LB-3 shown in SEQ ID NO. 11 is connected with a quenching group, and the 3' end is connected with a fluorescent group.
Alternatively, the fluorophore is AMCA, pacific Blue, atto 425, BODIPY FL, FAM, alexa Fluor 488, TET, JOE, yakima Yellow, VIC, HEX, quasar 570, cy3, NED, TAMRA, ROX, aquaPhuor 593, texas Red, atto 590, cy5, quasar 670, or Cy5.5.
Alternatively, the quenching group is BHQ1, BHQ2, BHQ3, dabcyl, eclipse, or MGB.
Specifically, the 5 'end of the loop primer LF shown in SEQ ID NO. 5 is connected with BHQ2, and the 3' end is connected with Cy5.
Specifically, the 5 'end of the loop primer LB-3 shown in SEQ ID NO. 11 is connected with BHQ2, and the 3' end is connected with VIC.
In view of the fact that the LAMP primer group can be used for realizing specific detection of the human circovirus. Therefore, the application also claims the application of the LAMP primer group in preparing a human circovirus detection reagent or kit.
The application also provides an LAMP kit for detecting the human circovirus, which contains primers shown in SEQ ID NO. 1-11.
Specifically, the kit further comprises: sample lysate, positive control, negative control, and reagents required for isothermal amplification reaction.
Optionally, the negative control is sterile water.
Alternatively, the positive control is a recombinant plasmid containing a HuCV-1CAP gene sequence, designated as puc-HuCV-1; and a recombinant plasmid containing the sequence of the HuCV-2CAP gene, designated as puc-HuCV-2.
Specifically, the reagents required for the isothermal amplification reaction include: neb Q5 high-fidelity enzyme with chainSubstitution-ability DNA polymerase, dNTP, mgSO 4 And 10 Xbuffer buffer.
Specifically, the 10×isothermal amplification buffer contains the following concentrations of each component: 100mM Tris-HCl (pH 8.5), 500nM KCl, 15nM MgCl 2 。
Specifically, the DNA polymerase is Bst DNA polymerase.
Specifically, the kit also contains a color development liquid.
Optionally, the color development liquid is phenol red, neutral red or cresol purple.
The application also provides a method for rapidly identifying the LAMP result, which comprises the steps of adding a color development liquid into the LAMP reaction product, and if the color development liquid shows that the LAMP reaction product is alkaline, indicating that the detection result is positive. The principle is that in the process of synthesizing DNA by LAMP reaction, the released byproducts comprise pyrophosphate and H + Therefore, the pH value of the reaction solution changes along with the progress of the reaction, the initial alkalinity changes into acidity, and the visual judgment of the amplification result is realized by using a pH sensitive indicator as a dye to detect DNA amplification, so that positive and negative results are distinguished.
Specifically, the LAMP reaction system prepared by using the kit disclosed by the application comprises the following steps: taking 25. Mu.L of isothermal amplification system as an example, 2.5. Mu.L of 10 Xisothermal amplification system buffer, 1. Mu.L of 8U/. Mu.L of Bst DNA polymerase, 100mM MgS0 4 1. Mu.L, 10mM dNTP 3.5. Mu.L, 10. Mu.M primer F3.5. Mu.L, 10. Mu.M primer B3.5. Mu.L, 80. Mu.M primer FIP 0.5. Mu.L, 80. Mu.M primer BIP 0.5. Mu.L, 40. Mu.M primer LF 0.5. Mu.L, 40. Mu.M LB primer 0.5. Mu.L, template 3. Mu.L, no nuclease water up to 25. Mu.L.
If the primer group does not contain the primer LF, the components are not added when the reaction system is prepared, and the reaction system is complemented by water without the nuclease.
Specifically, the isothermal amplification reaction conditions of LAMP were: reacting for 10-60 min at 50-65 ℃.
Preferably, the optimal reaction conditions are: the reaction was carried out at 65℃for 50min.
The application has the following beneficial effects:
the application provides an LAMP primer group and a kit for detecting human circovirus. By utilizing the LAMP primer group disclosed by the application, the typing and common detection of the type 1 and the type 2 of the human circovirus can be realized, and the detection process only needs isothermal amplification, does not need expensive amplification instruments and complex amplification procedures like PCR, can finish the detection within 50min, and has the advantages of high efficiency and high speed. Meanwhile, the LAMP primer group has good specificity and high sensitivity when being used for detecting the human circovirus, the detection limit is at least 1 copies/mu L, and the concentration above 10 copies/mu L can be stably detected, so that the sensitive, specific and accurate detection of the human circovirus is realized. In addition, the application also provides a kit for detecting the human circovirus, the detection of the human circovirus can be realized in a short time by using the kit, the operation is simple, the result is easy to judge, and the rapid detection of the human circovirus is facilitated.
Drawings
FIG. 1 shows the sensitivity test results of isothermal amplification of the HuCV-2CAP gene with the CAP2-4-1 primer set.
FIG. 2 shows the sensitivity test results of 5 LAMP primer groups designed for the HuCV-1CAP gene in Table 1.
FIG. 3 shows the sensitivity test results of 9 LAMP primer groups designed for the HuCV-2CAP gene in Table 2.
FIG. 4 shows the sensitivity test results of isothermal amplification of the HuCV-1CAP gene with the CAP1-2 primer set.
FIG. 5 shows the sensitivity test results of isothermal amplification of the HuCV-2CAP gene with the CAP2-4-3 primer set.
FIG. 6 shows the sensitivity test results of isothermal amplification of the HuCV-1CAP gene by combining the CAP1-2 primer set with the CAP2-4-3 primer set.
FIG. 7 shows the sensitivity test results of isothermal amplification of the HuCV-2CAP gene by combining the CAP1-2 primer set with the CAP2-4-3 primer set.
FIG. 8 shows the results of CAP-1-2 primer sets for amplifying the CAP genes of HuCV-1 and HuCV-2, respectively.
FIG. 9 shows the results of CAP-2-4-3 primer sets for amplifying the CAP genes of HuCV-1 and HuCV-2, respectively.
FIG. 10 shows the results of specific detection of isothermal amplification of 8 common viruses by CAP1-2 and CAP2-4-3 primer sets.
Detailed Description
The application is further illustrated in the following drawings and specific examples, which are not intended to limit the application in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 LAMP primer design
According to the application, a plurality of groups of specific LAMP primer groups are respectively designed according to two typed CAP genes of HuCV-1 (accession number GenBank: ON 677309) and HuCV-2 (accession number GenBank: ON 226770) reported in NCBI, and a plurality of groups with better pre-experiment effect are selected from the groups; wherein, the primer group for detecting HuCV-1 comprises: CAP-1-1, CAP-1-2, CAP-1-3, CAP-1-4, and CAP-1-5; the primer set for detecting HuCV-2 comprises: CAP-2-1, CAP-2-2, CAP-2-3, CAP-2-4-1, and CAP-2-5. As found in the early experiments, the CAP-2-4-1 primer set had better amplification but insufficient specificity. Therefore, the application optimizes the LB primer sequence in the CAP-2-4-1 primer group, designs CAP-2-4-2, CAP2-4-3, CAP2-4-4 and CAP2-4-5 primer groups respectively, wherein the F3, B3, FIP and BIP primers are consistent with the primer sequences in the CAP-2-4-1 primer groups, and only the LB sequences are different. Specific sequences (5 '-3') of the primers are shown in tables 1 and 2, respectively:
TABLE 1 LAMP primer set for detection of HuCV-1 Virus
TABLE 2 LAMP primer sets for detection of HuCV-2 Virus
When the LAMP primer group is used for detection, the 5 'end of one of the loop primers needs to be connected with a quenching group, and the 3' end needs to be connected with a fluorescent group.
Alternatively, the fluorophore is AMCA, pacific Blue, atto 425, BODIPY FL, FAM, alexa Fluor 488, TET, JOE, yakima Yellow, VIC, HEX, quasar 570, cy3, NED, TAMRA, ROX, aquaPhuor 593, texas Red, atto 590, cy5, quasar 670, or Cy5.5. The quenching group is BHQ1, BHQ2, BHQ3, dabcyl, eclipse or MGB.
Example 2 screening of LAMP primer group
The concentrations of plasmids puc-HuCV-1 and puc-HuCV-2 (synthesized by general biological company) containing the target gene of interest (CAP gene) were measured by a NanoDrop One/OneC micro nucleic acid protein concentration meter, respectively. Puc-HuCV-2 was diluted to 1000 copies/. Mu.L, 100 copies/. Mu.L, 10 copies/. Mu.L, and 1 copies/. Mu.L in a 10-fold gradient, and LAMP isothermal amplification reactions were performed with CAP2-4-1 primer sets using all diluted samples as templates, with 2 replicates for each reaction. The LAMP isothermal amplification reaction system is shown in Table 3, and the LAMP isothermal amplification reaction conditions are as follows: reacting for 10-60 min at 50-65 ℃. The optimal reaction conditions were tested: the reaction was carried out at 65℃for 50min, and this reaction condition was used in this example.
TABLE 3LAMP Single primer set isothermal amplification reaction System
| Sequence number | Component (A) | Concentration/. Mu.L |
| 1 | 10X isothermal amplification buffer | 2.5 |
| 2 | 100mM MgSO 4 | 2 |
| 3 | 10mM dNTP | 3.5 |
| 4 | 10 mu M primer F3 | 0.5 |
| 5 | 10 mu M primer B3 | 0.5 |
| 6 | 80 mu M primer FIP | 0.5 |
| 7 | 80 mu M primer BIP | 0.5 |
| 8 | 20 mu M primer LB | 0.5 |
| 9 | 20 mu M primer LF | 0.5 |
| 10 | 8U Bst polymerase | 1 |
| 11 | NEB Q5 high-fidelity enzyme | 0.075 |
| 12 | Template | 3 |
| 13 | Nuclease-free water | To 25 mu L |
No. 8 (LB primer) and No. 9 (LF primer) are not all primer sets, and the reaction system without No. 8 (LB primer) and No. 9 (LF primer) components is replaced by the amount of nuclease-free water, namely, the reaction system is complemented to 25 mu L by the nuclease-free water.
Specifically, the 10×isothermal amplification buffer contains the following concentrations of each component: 100mM Tris-HCl (pH 8.5), 500nM KCl, 15nM MgCl 2 。
The sensitivity detection results of isothermal amplification of the HuCV-2CAP gene by the CAP2-4-1 primer set are shown in FIG. 1. As can be seen from FIG. 1, the CAP-2-4-1 primer set was specific for 1 copies/. Mu.L (10 0 copies/. Mu.L) concentration templates all have amplification signals, the sensitivity is higher, but the amplification curve of negative control (NTC) also has signals, which indicates that the specificity of the corresponding primer group of CAP-2-4-1 is not high. Therefore, the LB primer sequence of CAP-2-4-1 is optimized, and LB-2, LB-3, LB-4 and LB5 loop primers are designed to be combined with the F3 primer, B3 primer, FIP primer and BIP primer of CAP-2-4-1 to form CAP-2-4-2, CAP-2-4-3, CAP-2-4-4 and CAP-2-4-5 primer groups.
The 5 LAMP primer groups for detecting HuCV-1 and the 9 LAMP primer groups for detecting HuCV-2 (14 groups in total, as shown in tables 1 and 2, respectively) are preferable. Wherein, the puc-HuCV-1 plasmid is used as an amplification template of the HuCV-1 virus detection primer group, the puc-HuCV-2 plasmid is used as an amplification template of the HuCV-2 virus detection primer group, and the template concentration is unified to be 1000 copies/. Mu.L. The LAMP isothermal amplification reaction system is shown in Table 3, and the LAMP isothermal amplification reaction conditions are 65℃for 50min. Amplification efficiencies were compared between the primer sets under equivalent conditions.
The sensitivity test results of 5 LAMP primer groups designed for the HuCV-1CAP gene in Table 1 are shown in FIG. 2. As can be seen from FIG. 2, the CAP-1-2 primer set exhibited the shortest amplification curve time compared to the other primer sets at the same template concentration. The sensitivity test results of 9 LAMP primer sets designed for the HuCV-2CAP gene in Table 2 are shown in FIG. 3, and it is clear from FIG. 3 that no NTC jump occurs in the CAP-2-4-3 primer set after the loop primer sequence is changed, indicating that the CAP-2-4-3 primer set has strong specificity and rapid amplification signal, so that the CAP-2-4-3 primer set is finally selected. CAP-1-2 and CAP-2-4-3 were used as the preferred primer sets for subsequent experiments.
The application also provides a kit for detecting the human circovirus, which comprises a primer group CAP-1-2 and/or CAP-2-4-3, wherein the nucleotide sequence of the primer contained in the primer group CAP-1-2 is shown as SEQ ID NO. 1-6; the nucleotide sequence of the primer contained in the primer group CAP-2-4-3 is shown in SEQ ID NO. 7-11. In addition to the above primer set, the kit further comprises 10×isothermal amplification system buffer, 8U/. Mu.L Bst DNA polymerase, 100mM MgS0 4 10mM dNTPs, sample lysates, positive controls (puc-HuCV-1 and puc-HuCV-2 plasmids), negative controls (sterile water or nuclease-free water).
Example 3 sensitivity detection of Single tube Single target CAP1-2 primer set and CAP2-4-3 primer set
After the primers are preferred, the concentration of the puc-HuCV-1 plasmid and the puc-HuCV-2 plasmid are measured by using a NanoDrop One/OneC micro nucleic acid protein concentration measuring instrument, and then diluted into 1000 copies/. Mu.L, 100 copies/. Mu.L, 10 copies/. Mu.L and 1 copies/. Mu.L by 10-fold gradient, and the LAMP detection kit provided by the application is used for amplification by taking all the diluted samples as templates, and 2 repeats are set for each reaction.
The LAMP amplification system was referred to the LAMP isothermal amplification reaction system of Table 3 in example 2.
The reaction conditions are as follows: the reaction is carried out for 50min at 65 ℃, namely, single-tube single-target reaction is carried out, namely, a single-tube reaction system only contains the puc-HuCV-1 plasmid or the puc-HuCV-2 plasmid and the CAP1-2 primer group or the CAP2-4-3 primer group corresponding to the puc-HuCV-1 plasmid or the puc-HuCV-2 plasmid.
The detection results of the sensitivity of the isothermal amplification of the HuCV-1CAP gene by the CAP1-2 primer set and the detection results of the sensitivity of the isothermal amplification of the HuCV-2CAP gene by the CAP2-4-3 primer set are shown in FIG. 4 and FIG. 5, respectively. As can be seen from FIGS. 4 and 5, the negative control detection results are normal, no fluorescent signal exists, and the positive sample has a fluorescent signal, which indicates that the primer set of the application can realize the detection of HuCV-1 or HuCV-2. According to the combination of the detection results of 2 times of repetition, the probability detection of the primer group to 1 copies/mu L is proved, all DNA templates with the other concentrations are detected, and the concentration above 10copies/reaction can be stably detected according to the conversion of the sample adding volume of 3 mu L.
Example 4 sensitivity detection of Single tube double target CAP-1-2 primer set and CAP-2-4-3 primer set
The primers CAP-1-2 and CAP-2-4-3 were placed in the same reaction tube for isothermal amplification using the puc-HuCV-1 plasmid and puc-HuCV-2 plasmid as templates, which were diluted with 10-fold gradients to 1000 copies/. Mu.L, 100 copies/. Mu.L, 10 copies/. Mu.L, and 1 copies/. Mu.L, respectively, to see if the combined use of the two primer sets would have an effect on the detection sensitivity for detecting human circovirus type 1 or type 2, and 2 replicates were used for each reaction. The LAMP isothermal amplification reaction system is shown in Table 4.
TABLE 4 LAMP double primer group isothermal amplification reaction system
The detection result of the sensitivity of the isothermal amplification of the HuCV-1CAP gene by the CAP1-2 primer group and the CAP2-4-3 primer group is shown in FIG. 6, and the detection result of the sensitivity of the isothermal amplification of the HuCV-2CAP gene by the CAP1-2 primer group and the CAP2-4-3 primer group is shown in FIG. 7. As can be seen from FIGS. 6 and 7, the concentration of detectable target in the CAP-1-2 primer set was 1 copies/. Mu.L, and the concentration of detectable target in the CAP-2-4 primer set was 1 copies/. Mu.L, indicating that the combination of CAP-1-2 primer set and CAP-2-4-3 primer set did not affect the sensitivity, and that CAP-1-2 and CAP-2-4-3 primer set together detected human circovirus types 1 and 2.
Example 5 Single tube Single target CAP-1-2 primer set and CAP-2-4-3 primer set specific detection
In order to detect the detection specificity of the CAP-1-2 primer set and the CAP-2-4-3 primer set, the present example uses the CAP-1-2 primer set to amplify the puc-HuCV-1 plasmid and the puc-HuCV-2 plasmid, and the CAP-1-2 primer set also amplifies the puc-HuCV-1 plasmid and the puc-HuCV-2 plasmid, respectively, and the amplification system and conditions are the same as in example 2.
The results of the amplification reactions of the CAP-1-2 primer set on the HuCV-1CAP gene and the HuCV-2CAP gene, respectively, are shown in FIG. 8, and the results of the amplification reactions of the CAP-2-4-3 primer set on the HuCV-1CAP gene and the HuCV-2CAP gene, respectively, are shown in FIG. 9. As shown in FIGS. 8 and 9, the CAP1-2 primer set only amplifies the puc-HuCV-1 plasmid template, and the CAP2-4-3 primer set only amplifies the puc-HuCV-2 plasmid template, and no amplified signal is detected after cross detection, which indicates that the CAP1-2 primer set and the CAP2-4-3 primer set have high specificity, and can realize the typing detection of two human circoviruses.
Example 6 specific detection of different viruses by single tube double target CAP-1-2, CAP-2-4-3 primer sets
In order to detect the detection specificity of the CAP-1-2 primer set and the CAP-2-4-3 primer set to other common human viruses, the embodiment uses 8 common human viruses of DNA, the puc-HuCV-1 plasmid and the puc-HuCV-2 plasmid as templates for detection; wherein, the addition amount of each template was 1. Mu.L, 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, HIV type 1, hepatitis B virus, hepatitis C virus and hepatitis G virus.
The results of specific assays for CAP1-2 and CAP2-4-3 primer sets for amplification of 8 common viruses are shown in FIG. 10. As is clear from FIG. 10, other viral DNAs except for the amplified signals of HuCV-1 and HuCV-2 did not detect the amplified signals, indicating that the specificity of the primer set of CAP-1-2 and CAP-2-4-3 was high, and human circovirus could be specifically detected. In addition, the application also compares the CAP1-2 and CAP2-4-3 primer groups with CAP genes in PCV, and the result shows that the interval anchored by the CAP1-2 and CAP2-4-3 primer groups has huge difference in the pairing rate with the CAP genes in PCV, namely, the CAP1-2 and CAP2-4-3 primer groups can not amplify PCV and have high specificity to human circovirus.
In conclusion, the LAMP detection primer group, the kit and the detection method for the HuCV-1 and HuCV-2 human circovirus provided by the application can finish the detection of the novel human circovirus within 50 minutes, have high detection speed and simple and convenient operation, and can be widely applied to the rapid differential diagnosis of the novel human circovirus.
The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present application should be made in the equivalent manner, and the embodiments are included in the protection scope of the present application.
Claims (10)
1. The LAMP primer group for detecting the human circovirus type 1 is characterized by 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.
2. The LAMP primer group for detecting the human circovirus type 2 is characterized by 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.
3. A LAMP primer set for detecting human circovirus, comprising the LAMP primer set of claims 1 and 2.
4. The LAMP primer group according to any one of claims 1 to 3, wherein 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 attached to the 5 'end and a fluorescent group attached to the 3' end.
5. The LAMP primer group according to claim 4, wherein 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, aquar Phuor 593, texas Red, atto 590, cy5, quasar 670 or Cy5.5.
6. The LAMP primer set of claim 4, wherein the quenching group is BHQ1, BHQ2, BHQ3, dabcyl, eclipse or MGB.
7. The use of the LAMP primer group according to any one of claims 1 to 6 in the preparation of a human circovirus detection reagent or kit.
8. A LAMP kit for detecting human circovirus, characterized by comprising the LAMP primer group according to any one of claims 1 to 6.
9. The LAMP kit of claim 8, further comprising the following components: sample lysate, positive control, negative control, and reagents required for isothermal amplification reaction.
10. The LAMP kit of claim 9, wherein the isothermal amplification reaction is performed byThe reagent comprises the following components: neb Q5 high-fidelity enzyme, DNA polymerase, dNTP, mgSO 4 And 10 Xbuffer buffer.
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