CN117070673B - LAMP detection primer group for pangolin alpha coronavirus and application thereof - Google Patents
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Abstract
The invention relates to an LAMP detection primer group for pangolin alpha coronavirus and application thereof, and relates to the technical field of virus detection. The LAMP detection primer group comprises 1 pair of inner primers, 1 pair of outer primers and a loop primer LB; the inner primer comprises an upstream inner primer FIP and a downstream inner primer BIP, and the outer primer comprises an upstream outer primer F3 and a downstream outer primer B3. The primer group can realize specific, stable, sensitive and efficient detection of the alpha coronavirus of the pangolin source, can be applied to the field detection of a base layer, and has important value in the aspect of the detection of the alpha coronavirus.
Description
Technical Field
The invention relates to the technical field of virus detection, in particular to an LAMP detection primer group for pangolin alpha coronavirus and application thereof.
Background
Coronaviruses are enveloped single-stranded positive-strand, non-segmented RNA viruses that resemble coronaviruses, so they are named coronaviruses, which are very diverse, a considerable variety of which have the property of cross-species transmission, and are a hotspot in medical and animal medicine research. According to the current nomenclature of the International Commission on viral classification, the coronaviridae is divided into 4 genera, alpha, beta, gamma (corresponding to the previous 1,2 and 3 groups, respectively) and Delta coronaviruses, and new genera and new species are now found. Beta coronaviruses are a major concern and have strong cross-species transmission properties, while recent studies have found that some species of alpha coronaviruses also have cross-species transmission properties, such as canine alpha coronaviruses. Alpha coronavirus of pangolin scales is a newly discovered virus, and sequence analysis shows that the virus also has the possibility of cross-species transmission.
Pangolin is a living animal, the immune system is more special, many gene sequences related to genome immunity are pseudogenes, and are easy to infect various pathogenic microorganisms, such as Epikovia, parainfluenza virus, coronavirus, parvovirus and the like, and living or foraging cavities of pangolin not only have activity, but also can have more than 70 animals such as bat, porcupine, weasel, bamboo rat, snake and the like to directly or indirectly utilize the cavities, so that pangolin is likely to infect or spread various pathogenic microorganisms, and is a storage host for pathogens of some human and animal comorbicular diseases. The existing research shows that pangolin can be infected with BETA coronavirus and also can be infected with alpha coronavirus, and through sequence analysis, the coronavirus carried by pangolin has a certain cross-species transmission capability, has a certain potential hazard to human, and has extremely important value for research on tracing, evolution, variation, cross-species transmission and the like of the alpha coronavirus. By reporting and analyzing coronaviruses of different animals, it is inferred that the types of the alpha coronaviruses carried by the animals are more, the transmission path is more complex and wider than that of our cognition, so that a faster and simpler detection method is needed to detect alpha coronaviruses of more species in a larger range.
Disclosure of Invention
Aiming at the problems, the invention provides the LAMP detection primer group for the pangolin alpha coronavirus, and the primer group can realize specific, stable, sensitive and efficient detection of the pangolin alpha coronavirus, can be applied to the field detection of a base layer, and has important value in the aspect of the detection of the alpha coronavirus.
The invention provides an LAMP detection primer group for pangolin alpha coronavirus, which comprises 1 pair of inner primers, 1 pair of outer primers and a loop primer LB; the inner primers comprise an upstream inner primer FIP and a downstream inner primer BIP, and the outer primers comprise an upstream outer primer F3 and a downstream outer primer B3;
upstream inner primer FIP: CAAAATCCTTCCTGCTTTTAACACGGATATTGCTTTAATGACAGGCTAA (SEQ ID NO: 1);
downstream inner primer BIP: ATCAGCCAATATCAGAAAAGTGAGTAATTCCTCGGAACGACCA (SEQ ID NO: 2);
upstream outer primer F3: ACTGTCCATACCATACACATG (SEQ ID NO: 3);
downstream outer primer B3: TACAAGAGTAGACAGCGC (SEQ ID NO: 4);
loop primer LB: TGTAAGGCAACCCGATGTTTAAAAC (SEQ ID NO: 5).
The loop-mediated isothermal amplification (LAMP) technology has higher specificity and sensitivity, is simple to use and does not need special equipment. LAMP was performed by BstDNA polymerase having strand displacement activity (i.e., modified DNA polymerase from Bacillus stearothermophilus) at a temperature ranging from 61℃to 69℃and under isothermal conditions for 30-60 min. The final amplified product is a mixture of stem-loop DNA with different stems, which can be analyzed by direct observation or by agarose gel. The LAMP method has been developed for detecting a variety of viruses in animals, including newcastle disease virus, avian influenza virus, peste des petits ruminants virus, and West Nile virus, but has not been applied to the detection of pangolin-derived alpha coronaviruses. Therefore, the inventor proposes to analyze the whole genome sequence of Pan-alpha-Cov to obtain an alpha coronavirus conservation region sequence with the length of 824bp, and design the LAMP detection primer group aiming at the conservation region sequence, wherein the amplification target sequence of the LAMP detection primer group is between 414 and 601, and meanwhile, the LAMP detection primer group is combined with a loop-mediated isothermal amplification technology to realize the specific, stable, sensitive and efficient detection of alpha coronavirus of pangolin sources.
In one embodiment, the molar ratio of the outer primer, the inner primer and the loop primer LB is (3.5-4.5): 1: (1.8-2.2).
In one embodiment, the molar ratio of the outer primer, the inner primer, and the loop primer LB is 4:1:2.
the invention also provides a kit for detecting the pangolin alpha coronavirus, and an amplification reaction system of the kit comprises the LAMP detection primer group.
In one embodiment, the amplification reaction system of the kit further comprises: indicator, bstDNA polymerase.
The indicator is added before the reaction, so that the pollution is small, and the detection result is visual.
In one embodiment, the indicator is HNB.
Mg 2+ Combined with HNB to make the initial color of the reaction system be violet, mg as the reaction proceeds 2+ React with the separated pyrophosphate ions to generate magnesium pyrophosphate precipitate, and hydroxy naphthol blue loses Mg 2+ So that the system color becomes sky blue, and the unreacted system still keeps violet, thereby judging the LAMP reaction result. The detection result can be monitored by naked eyes in real time, the cover is not required to be opened, electrophoresis is not required, and the method is very convenient.
In one embodiment, the amplification reaction system of the kit comprises: an indicator, bstDNA polymerase, buffer, water and the LAMP detection primer set.
In one embodiment, the amplification reaction system of the kit comprises: 10 Xreaction buffer 2.5.+ -. 0.5. Mu.L, 40. Mu.M upstream inner primer FIP, 40. Mu.M downstream inner primer BIP, 10. Mu.M upstream outer primer F3, 10. Mu.M downstream outer primer B3, 20. Mu.M loop primer LB 1.+ -. 0.2. Mu.L each, bst II DNA Polymerase (8000U/mL) 1.+ -. 0.2. Mu.L, HNB 2.0.+ -. 0.2. Mu.L, 100mM MgSO 4 1.5+ -0.2 μL,10mM dNTP 3.5+ -0.2 μL,10mM betaine 2+ -0.2 μL, ultrapure 6.5+ -1 μL water.
The invention also provides a LAMP detection method of pangolin alpha coronavirus with non-diagnostic purpose, which comprises the following steps: extracting nucleic acid of a sample to be detected, carrying out reverse transcription, and carrying out isothermal amplification by adopting the kit.
The LAMP detection method has high specificity, does not cross react with other viruses, has the lowest detection limit of 146.5 copy/mu L, can realize the instant detection of the viruses by applying the detection method to the detection of the pangolin alpha coronavirus, has high detection sensitivity and specificity, low cost of manpower and equipment and short period, does not depend on a laboratory, and is particularly suitable for on-site detection. The rapid detection technology can be popularized and applied to epidemiological investigation and epidemic situation monitoring of pangolin alpha coronavirus, and has good practical significance and broad market prospect.
In one embodiment, the isothermal amplification comprises incubation, the conditions of which: incubating at 64-66 ℃ for 55-65min.
In one embodiment, the isothermal amplification further comprises a reaction stop, the conditions of which are: and water bath at 75-85deg.C for 4-6min.
The invention also provides application of the LAMP detection primer group in developing and/or preparing a product with the application of diagnosing and/or prognosis evaluating diseases caused by infection of pangolin alpha coronaviruses.
The invention also provides application of the kit in developing and/or preparing a product with diagnostic and/or prognostic evaluation purposes for pangolin alpha coronavirus infection.
Compared with the prior art, the invention has the following beneficial effects:
the LAMP detection primer group for the pangolin alpha coronavirus can realize specific, stable, sensitive and efficient detection of the pangolin alpha coronavirus, and the detection of the pangolin alpha coronavirus by adopting the LAMP detection primer group is determined by adopting a method of continuously diluting a positive standard product by 10 times, wherein the minimum detection limit can reach 146.5 copy/mu L, and the sensitivity is 10 times compared with that of a conventional fluorescence quantitative PCR method; the LAMP detection primer group is used for detecting pangolin beta coronavirus (Pan-beta-Cov), canine coronavirus (CCov) and Feline Infectious Peritonitis Virus (FIPV), positive results are not found, the specificity is high, and the LAMP detection primer group can be applied to basic field detection and has important value in the aspect of alpha coronavirus detection.
Meanwhile, the LAMP detection method is simple and convenient to operate, does not need complex expensive instruments, is quick and efficient, can finish the judgment from the sample extraction to the result judgment within 60 minutes, has the timeliness equivalent to that of a TaqMan real-time fluorescent quantitative PCR method, is simple in reaction result judgment method, and can judge the result not only through agarose gel nucleic acid electrophoresis, but also through naked eyes under ultraviolet irradiation of fluorescent dye. The LAMP detection primer is combined with the LAMP amplification technology, so that the rapid and immediate detection of Pan-alpha-Cov can be realized, the defects of time consumption, labor consumption and high cost of Pan-alpha-Cov detection are overcome, the detection sensitivity and specificity are improved, the cost of labor and equipment is reduced, the detection period is shortened, and the rapid detection technology can be popularized and applied to epidemiological field real-time investigation and epidemic monitoring of Pan-alpha-Cov, and has good practical significance and broad market prospect.
Drawings
FIG. 1 is a graph showing the agarose electrophoresis result and the color indication change result of the LAMP method for detecting Pan- α -Cov, which were initially established in example 1, wherein the reaction temperature was 65℃and the reaction time was 60min, M in the electrophoresis chart on the left side of FIG. 1: DL2000,1 is positive control and NC is negative control; in the graph of the color indication change result on the right side of fig. 1, positive control and negative control are sequentially performed from left to right, and the result shows that the positive sample can be amplified to form a band, and the color of the HNB indicator is changed from purple to blue.
FIG. 2 is an agarose electrophoresis result and a color indication change result of the LAMP method of example 1 for optimizing the temperature of Pan- α -Cov, wherein M in the upper electrophoresis chart of FIG. 2: DL2000,1:59 ℃,2:61 ℃, 3:63 ℃, 4:65 ℃, 5:67 ℃, 6:69 ℃, NC: a negative control; in the color indication change result diagram below in fig. 2, 1 is shown in order from left to right: 59 ℃,2:61 ℃, 3:63 ℃, 4:65 ℃, 5:67 ℃, 6:69 ℃, NC: negative control.
FIG. 3 shows agarose electrophoresis results and color indication change results of the LAMP method for detecting Pan- α -Cov sensitivity test in example 2, wherein M in the upper electrophoresis chart of FIG. 3: DL2000,1: 146.5X10 7 copy/μL、2：146.5×10 6 copy/μL、3：146.5×10 5 copy/μL、4：146.5×10 4 copy/μL、5：146.5×10 3 copy/μL、6：146.5×10 2 copy/μL、7：146.5×10 1 copy/μL、8：146.5×10 0 copy/μL、9：146.5×10 -1 copy/. Mu.L; in the color indication change result diagram under FIG. 3, 146.5X10 from left to right 7 copy/μL、2：146.5×10 6 copy/μL、3：146.5×10 5 copy/μL、4：146.5×10 4 copy/μL、5：146.5×10 3 copy/μL、6：146.5×10 2 copy/μL、7：146.5×10 1 copy/μL、8、146.5×10 0 copy/μL、9：146.5×10 -1 copy/μL。
FIG. 4 is a graph showing the results of a sensitivity test for detecting Pan- α -Cov by q-PCR in example 2, wherein A to H are sequentially combined with 146.5X10 7 ～146.5×10 0 copy/. Mu.L 8 gradients correspond.
FIG. 5 is a graph showing the results of a sensitivity test for detecting Pan- α -Cov by q-PCR in example 2, wherein A to H are sequentially combined with 146.5X10 7 ～146.5×10 0 copy/. Mu.L 8 gradients correspond.
FIG. 6 is a graph showing the results of the reproducibility of example 3, wherein M is DL2000, repeated in 1-10 batches, and the same sample is applied at the same time; 11-20 are the same samples, and are added every 2 days. The results show that the established RT-LAMP system has good intra-batch and inter-batch repeatability.
FIG. 7 is a graph showing the results of the specificity test in example 4, wherein samples 1,2, 5, 25 and 26 are subjected to specificity verification in example 4, cDNA (complementary deoxyribonucleic acid) numbers 25 and 26 are positive results, blue is visible by naked eyes, and agarose gel electrophoresis shows a characteristic trapezoid band; the detection results of other pathogens are negative, the visual observation is purple, and no characteristic trapezoid strip appears; wherein 25 is positive plasmid cDNA,26 is pangolin blood sample cDNA,1 is cat infectious peritonitis positive plasmid cDNA,2 is canine coronavirus positive plasmid cDNA,3 is pangolin beta coronavirus positive plasmid cDNA.
FIG. 8 is a graph showing the results of electrophoresis of example 1, comparative example 1 and comparative example 2, wherein 1 is comparative example 1,3 is example 1 and 5 is comparative example 2.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The reagents, materials and equipment used in the examples are all commercially available sources unless otherwise specified; the experimental methods are all routine experimental methods in the field unless specified.
Example 1
Designing an LAMP detection primer group and constructing an LAMP detection method.
1. And (3) preparing a Pan-alpha-Cov positive standard.
The genome sequence 824bp (shown as SEQ ID NO: 6) of the Pan-alpha-Cov is obtained by high-flux sequence detection, the full genome sequence of the Pan-alpha-Cov is obtained, the homology analysis is carried out by BLAST software, the RT-PCR verification is carried out, the genome sequence 824bp of the Pan-alpha-Cov is confirmed to be the alpha coronavirus, the target gene sequence is synthesized by a biological company, and meanwhile, the target gene sequence is connected with a vector, so that a positive standard substance is obtained.
GACATGGGAATTATTGGAAAAATTTGTTGGAAATACCTTATACATCACAACACCACAAGTACTTTCACTACCATTAGGTGCGGAAGTACGTTGTGATGATATTGAAGGATTCCATTGTTCTTGGCCAGGTTATAAAGATTATGCCCATGATCATGTTGATTTTCATTTTAATCCCTCTAATCCTTTCTATTCTTTTGTAGATACTTTCTATATTTCTTTAGGTGATAGACAGGATAAAATTTATCTTAGAGTTGTTGGTGCAACACCAAAAGAGAGAATGCTGACTATTGGTTGTCACACATCTTTCTCAGTAAACCTTCCAATTGGAACTCAGATTTACCATGACAAGGATATGCAACTTCTTGTCGAGGGAAAACATCTTGAGTGTTCTCATAGAGTTTACTTTGTGAAGTACTGTCCATACCATACACATGGATATTGCTTTAATGACAGGCTAAAGGTCTATGATCTGAAGCGTGTTAAAAGCAGGAAGGATTTTGAGAAAATCAGCCAATATCAGAAAAGTGAGTTGTAAGGCAACCCGATGTTTAAAACTGGTCGTTCCGAGGAATTACTGGTCATCGCGCTGTCTACTCTTGTACAGAATGGTAAGCACGTGTGATAGGAGGTACAAGCAACCCTATTGCATATTAGGAAGTTTAGATTTGATTTGGCAATGCTAGATTTAGTAATTTAGAGAAGTTTAAAGATCCGCTATGACGAGCCAACAATGGAAGAGCTAACGTCTGGATCTAGTGATTGTTTAAAATGTAAAATTGTTTGAAATTTTTTTTTTTTTTTTGTGTATCACTATCAAAAGGAAA(SEQ ID NO:6)；
2. Designing and synthesizing LAMP detection primer groups.
Designing a LAMP detection primer group aiming at the target gene sequence by using PrimerExplorer V4 software, and synthesizing primers, wherein the amplification target sequence of the LAMP detection primer group is between 414 and 601. The synthesized primers are screened, i.e., the synthesized primers are diluted and then subjected to primer screening, and amplified by conventional PCR. Finally, a set of primer group capable of specifically and sensitively detecting Pan-alpha-Cov is obtained after screening. The primer consists of an outer primer F3/B3, an inner primer FIP/BIP and a loop primer LB, and is specifically shown as follows.
Upstream inner primer FIP: CAAAATCCTTCCTGCTTTTAACACGGATATTGCTTTAATGACAGGCTAA (SEQ ID NO: 1);
downstream inner primer BIP: ATCAGCCAATATCAGAAAAGTGAGTAATTCCTCGGAACGACCA (SEQ ID NO: 2);
upstream outer primer F3: ACTGTCCATACCATACACATG (SEQ ID NO: 3);
downstream outer primer B3: TACAAGAGTAGACAGCGC (SEQ ID NO: 4);
loop primer LB: TGTAAGGCAACCCGATGTTTAAAAC (SEQ ID NO: 5).
3. Construction and optimization of the LAMP detection method.
1. And extracting nucleic acid of the sample to be tested.
According to the instructions of the TIANGEN company Magnetic Viral DNA/RNAkit nucleic acid extraction kit. Extracting RNA of a pangolin blood sample, carrying out reverse transcription to obtain cDNA, and carrying out detection by the LAMP method.
2. LAMP amplification reaction was performed.
The LAMP reaction system of this embodiment includes: 10 Xreaction buffer 2.5. Mu.L, 40. Mu.M upstream inner primer FIP, 40. Mu.M downstream inner primer BIP, 10. Mu.M upstream outer primer F3, 10. Mu.M downstream outer primer B3, 20. Mu.M loop primer LB 1. Mu.L each, bst II DNA Polymerase (8000U/mL) 1. Mu.L, HNB 2.0. Mu.L, 100mM MgSO 4 1.5. Mu.L, 10mM dNTP 3.5uL,10mM betaine 2. Mu.L, 1.0. Mu.L of cDNA and 6.5. Mu.L of ultrapure water.
The mixture was incubated at 59 ℃,61 ℃,63 ℃,65 ℃,67 ℃ and 69 ℃ for 60min to determine the optimal reaction temperature, and then after heat inactivation by 80 ℃ for 10min, the reaction was terminated. Sterile water was used as a negative control instead of template cDNA. If a color change is desired, HNB indicators are added prior to the reaction.
And (3) verifying results: LAMP product (10. Mu.L) was separated by 2.5% agarose gel electrophoresis and ethidium bromide stained for UV-visible results.
And (3) result judgment: the LAMP amplified pan- α -Cov agarose gel electrophoresed DNA product showed characteristic trapezoidal bands with multi-banded fragments, indicating that the final LAMP product was a mixture of stem-loop DNA of different stem lengths (FIG. 1). In contrast, the negative control lacks this characteristic multi-band ladder pattern.
Optimal reaction temperature and time optimization for LAMP detection of Pan-alpha-Cov: the optimal reaction temperature and time for Pan-alpha-CovLAMP detection were studied. The LAMP reaction at 65℃produced the sharpest and brighter bands than seen at the other temperatures (FIG. 2). Therefore, the optimal reaction condition for LAMP detection of Pan-alpha-Cov is 65℃for 60min
Example 2
Sensitivity experiments.
The Pan- α -Cov positive standard of example 1 was diluted 10-fold in gradient and used as a template for a total of 9 gradients (146.5X10 7 ～146.5×10 -1 copy/. Mu.L) was detected using the LAMP detection primer set of example 1 and optimal reaction conditions to determine the lowest detection limit of the detection method of example 1. Meanwhile, the following primer sequences are designed according to 824bp sequences of Pan-alpha-Cov:
q-PCR upstream primer Pan-alpha-Cov-F: 5'-CCCGATGTTTAAAACTGGTCGT-3' (SEQ ID NO: 7);
q-PCR downstream primer Pan- α -Cov-R:5'-CTTCCATTGTTGGCTCGTCA-3' (SEQ ID NO: 8).
Detection by q-PCR method was compared with the LAMP method of example 1.
The results are shown in FIGS. 3, 4, 5 and the following table, 10-fold serial dilutions of Pan- α -Cov positive plasmid (from 10 7 To 10 -1 copy/. Mu.L) was used to determine sensitivity, and the positive reaction mixture exhibited a typical trapezoidal pattern. In triplicate, 100% of samples containing 146.5 copy/. Mu.L of LAMP positive plasmid were detected, which was higher than the established q-PCR sensitivity. The LAMP established by the invention has better sensitivity to Pan-alpha-Cov.
TABLE 1 sensitivity test results of q-PCR method detection
Example 3
And (5) repeating the experiment.
The LAMP detection primer group and the optimal reaction conditions in the embodiment 1 are adopted for repeated detection, and the same sample is added at the same time; the same sample was applied every 2 days.
As shown in FIG. 6, the results of the LAMP repeatability analysis show that the LAMP established by the invention has good intra-batch and inter-batch repeatability for Pan-alpha-Cov.
Example 4
Specificity experiments.
The canine coronavirus (CCov) nucleic acid, feline Infectious Peritonitis Virus (FIPV) nucleic acid, pangolin alpha coronavirus Pan-alpha-Cov nucleic acid, pangolin beta coronavirus Pan-beta-Cov nucleic acid used in this example were identified and stored by the wild animal microbiology laboratory in zoo Guangzhou.
The LAMP detection primer set and the optimal reaction conditions of example 1 are adopted for specific detection, and samples to be detected comprise canine coronavirus (CCov) nucleic acid, feline Infectious Peritonitis Virus (FIPV) nucleic acid, pangolin beta coronavirus (Pan-beta-Cov) nucleic acid, pan-alpha-Cov positive standard and negative control.
As a result, as shown in FIG. 7, the results of LAMP-specific analysis revealed that no amplification products were observed in the reaction mixtures containing CCov, FIPV, pan-. Beta. -CovcDNA templates, respectively, indicating that LAMP established in the present invention had high specificity for Pan-. Alpha. -Cov.
Example 5
Detection of clinical samples.
Nucleic acid extraction and reverse transcription were performed on 22 samples of pangolin tissue taken from fine, according to commercial kits, and the viral RNA extraction kit specifically used in this example was purchased from AXYGEN, and the reverse transcription kit was purchased from TaKaRa.
The detection was performed according to the q-PCR method established in example 2, compared with the LAMP method of example 1.
The results show that: the LAMP method of example 1 detects that 6 samples are positive, the positive rate is 27.3%, and then the virus separation and identification prove that the 6 samples are positive samples; the q-PCR method detects 4 positive samples, and the positive rate is 18.3%. Thus confirming the higher sensitivity and accuracy of the LAMP detection of example 1.
Comparative example 1
Substantially the same as in example 1 (LAMP detection primer group and optimal reaction conditions of example 1) except that the LAMP detection primer group of this comparative example is as follows:
F3-2：ACTGTCCATACCATACACATG(SEQ ID NO:9)
B3-2：TACAAGAGTAGACAGCGC(SEQ ID NO:10)
FIP-2：CAAAATCCTTCCTGCTTTTAACACGGATATTGCTTTAATGACAGGCTAA(SEQ ID NO:11)
BIP-2：ATCAGCCAATATCAGAAAAGTGAGTAATTCCTCGGAACGACCA(SEQ ID NO:12)
LB-2：TGTAAGGCAACCCGATGTTTAAA(SEQ ID NO:13)
as shown in lane 1 of FIG. 8, the band clearly became clear in comparison with lane 3 (LAMP detection primer group and optimal reaction conditions of example 1).
Comparative example 2
Substantially the same as in example 1 (LAMP detection primer group and optimal reaction conditions of example 1) except that the LAMP detection primer group of this comparative example is as follows:
F3-3：GTGTTAAAAGCAGGAAGGATT(SEQ ID NO:14)
B3-3：TTGCCAAATCAAATCTAAACTTC(SEQ ID NO:15)
FIP-3：CGACCAGTTTTAAACATCGGGTGAGAAAATCAGCCAATATCAGA(SEQ ID NO:16)
BIP-3：TTCCGAGGAATTACTGGTCATCGATAGGGTTGCTTGTACCTC(SEQ ID NO:17)
LB-3：CGCTGTCTACTCTTGTACAGAATGG(SEQ ID NO:18)
as shown in FIG. 8, lane 5 shows the clearly lower band than lane 3 (LAMP detection primer set and optimal reaction conditions of example 1).
In conclusion, the LAMP detection primer group and the kit for pangolin alpha coronavirus can rapidly and accurately detect Pan-alpha-Cov, so that the defects of time and labor waste, detection cost and labor intensity are reduced, sensitivity and specificity are improved, and detection period is shortened in the existing Pan-alpha-Cov detection technology. The invention has the characteristics of quick and simple operation in scientific research and production practice, and is suitable for on-site instant detection.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. The LAMP detection primer group for the pangolin alpha coronavirus is characterized by comprising 1 pair of inner primers, 1 pair of outer primers and a loop primer LB; the inner primers comprise an upstream inner primer FIP and a downstream inner primer BIP, and the outer primers comprise an upstream outer primer F3 and a downstream outer primer B3;
upstream inner primer FIP: CAAAATCCTTCCTGCTTTTAACACGGATATTGCTTTAATGACAGGCTAA (SEQ ID NO: 1);
downstream inner primer BIP: ATCAGCCAATATCAGAAAAGTGAGTAATTCCTCGGAACGACCA (SEQ ID NO: 2);
upstream outer primer F3: ACTGTCCATACCATACACATG (SEQ ID NO: 3);
downstream outer primer B3: TACAAGAGTAGACAGCGC (SEQ ID NO: 4);
loop primer LB: TGTAAGGCAACCCGATGTTTAAAAC (SEQ ID NO: 5).
2. The LAMP-detecting primer set according to claim 1, wherein the molar ratio of the outer primer, the inner primer and the loop primer LB is (3.5-4.5): 1: (1.8-2.2).
3. A kit for detecting pangolin alpha coronavirus, characterized in that the amplification reaction system of the kit comprises the LAMP detection primer set as claimed in any one of claims 1 to 2.
4. The kit according to claim 3, wherein the amplification reaction system of the kit further comprises an indicator and BstDNA polymerase.
5. The kit according to any one of claims 3 to 4, wherein the amplification reaction system of the kit comprises: an indicator, bstDNA polymerase, buffer, water, and LAMP detection primer set of any one of claims 1-2.
6. A LAMP detection method of pangolin alpha coronavirus with non-diagnostic purpose is characterized by comprising the following steps: extracting nucleic acid of a sample to be detected, carrying out reverse transcription, and carrying out isothermal amplification by using the kit according to any one of claims 3 to 5.
7. The LAMP detection method of claim 6, wherein the isothermal amplification comprises incubation, and wherein the conditions of the incubation are: incubating at 64-66 ℃ for 55-65min.
8. The LAMP detection method as claimed in claim 7, wherein the isothermal amplification further comprises a reaction stop, and the reaction stop conditions are as follows: and water bath at 75-85deg.C for 4-6min.
9. Use of the LAMP detection primer set of any one of claims 1-2 in the development and/or preparation of a product having diagnostic use for infection with pangolin alpha coronavirus.
10. Use of a kit according to any one of claims 3 to 5 for the development and/or preparation of a product having diagnostic use for infection with pangolin alpha coronavirus.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012024039A (en) * | 2010-07-26 | 2012-02-09 | Toshiba Corp | Nucleic acid primer set for lamp amplification for corona virus |
| CN115044710A (en) * | 2022-06-10 | 2022-09-13 | 广州动物园 | Primer group, kit and application for detecting pangolin beta coronavirus |
| CN116463461A (en) * | 2023-05-22 | 2023-07-21 | 厦门宝太生物科技股份有限公司 | Novel freeze-dried coronavirus detection kit based on visual RT-LAMP |
| WO2023159572A1 (en) * | 2022-02-28 | 2023-08-31 | 江苏汇先医药技术有限公司 | Detection kit, primer composition and detection method for novel coronavirus sars-cov-2 |
| CN116676429A (en) * | 2023-07-27 | 2023-09-01 | 广东省林业科学研究院 | LAMP primer group and method for detecting pangolin respiratory syncytial virus A and B |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012024039A (en) * | 2010-07-26 | 2012-02-09 | Toshiba Corp | Nucleic acid primer set for lamp amplification for corona virus |
| WO2023159572A1 (en) * | 2022-02-28 | 2023-08-31 | 江苏汇先医药技术有限公司 | Detection kit, primer composition and detection method for novel coronavirus sars-cov-2 |
| CN115044710A (en) * | 2022-06-10 | 2022-09-13 | 广州动物园 | Primer group, kit and application for detecting pangolin beta coronavirus |
| CN116463461A (en) * | 2023-05-22 | 2023-07-21 | 厦门宝太生物科技股份有限公司 | Novel freeze-dried coronavirus detection kit based on visual RT-LAMP |
| CN116676429A (en) * | 2023-07-27 | 2023-09-01 | 广东省林业科学研究院 | LAMP primer group and method for detecting pangolin respiratory syncytial virus A and B |
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