Whole genome capture primer combination of coxsackie A6, kit and application
By designing a combination of 7 pairs of primers in a stacked tiles, the segmented amplification of the entire genome of Coxsach A6 was solved, and the problem that the existing technology could not conduct whole genome research and typing identification was obtained, and high coverage and efficient full genome Genome amplification product, suitable for a variety of molecular biology experiments.
Patent Information
- Application Number
- CN202411372853.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2024-12-17
AI Technical Summary
The existing technology cannot conduct whole-genome research and typing identification of Coxsach A6, resulting in the inability to understand the genetic mutation and evolutionary relationship of the virus and the inability to effectively prevent and control the virus.
A combination of whole-genome capture primers for Coxsach A6 was designed, and 7 pairs of primers were designed using the tiled principle. The two primer pools were amplified in segments to ensure the integrity and coverage of the amplified products and achieve the whole-genome inversion. Transcription and enrichment amplification.
Accurate typing and full-length sequence acquisition of the Coxsach A6 subtype whole genome, with a coverage of up to 99%, and is suitable for various molecular biology experiments, including second-generation and third-generation whole genome sequencing.
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Figure CN119144767A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of primer detection, and in particular to a whole genome capture primer combination, a kit and an application of Coxsackie A6. Background Art
[0002] Hand, foot and mouth disease (HFMD) is a common infectious disease caused by a variety of enterovirus (EV) infections. The main pathogens include coxsackievirus A (CA) 4-7, 9, 10, 16 and B 1-3, 5, some serotypes of echovirus and EV71. HFMD is common in infants and young children aged 0-5 years. The typical manifestations are fever, maculopapular rash and herpes on the hands, feet, buttocks, mouth and other parts. Currently, CVA6 has surpassed EV71 and CVA16 to become the main pathogen of HFMD. The HFMD caused by coxsackievirus A6 has more extensive and more severe skin lesions. Vesicular bullous lesions can affect the back of the hands, back of the feet, calves, forearms, trunk, neck, and mouth. Severe symptoms lead to complications such as encephalitis and pulmonary hemorrhage that may be life-threatening.
[0003] CVA6 belongs to the Enterovirus genus of the Picornaviridae family, group A. It is icosahedral, non-enveloped, and single-stranded positive-strand RNA. The genome is about 7,400 bp long, including an open reading frame and 5′ and 3′ non-coding regions. CVA6 has the general physical and chemical properties of EV, and is resistant to low temperature and acid, but loses its activity at temperatures above 56°C and is sensitive to drying, ultraviolet light, and oxidants. CVA6 strains can be divided into six genotypes, from A to F, and the D genotype can be further subdivided into subtypes D1-3.
[0004] At present, there are mainly the following methods for the research and identification of CVA6: 1. Traditional serological method: collect blood samples from patients, separate serum and use specific antigens for detection. However, this method is time-consuming, virus isolation may fail, and the sensitivity is not enough; 2. Conventional quantitative PCR method: design primers and probes according to the characteristics of CVA6 nucleic acid sequence, and identify the presence and abundance of CVA6 by amplifying specific target fragments under specific reaction systems and reaction conditions. However, quantitative PCR can only identify the presence of Coxsackie CVA6 virus, and cannot perform further gene sequence analysis and typing; 3. Sequencing method: At present, the sequencing research on Coxsackie virus is mostly VP1 gene. VP1 gene is targeted for amplification and sequencing by designing primers, and the obtained sequence is analyzed by constructing evolutionary trees. For example, a five-poison fluorescent quantitative kit for detecting enterovirus provided by CN107513584B can only identify CVA6 virus but cannot perform whole genome research and typing identification.
[0005] In summary, there is currently no research on the whole genome of Coxsackie A6 on the market, which leads to an inability to fully understand the genetic variation and evolutionary relationships of the virus, and an inability to effectively prevent and control the virus. Summary of the invention
[0006] The purpose of the present invention is to provide a whole genome capture primer combination, a kit and an application of Coxsackie A6. A primer pool of 7 pairs of primers is designed according to the shingled principle for the whole genome of Coxsackie A6 subtype, and the primer pool is used to realize reverse transcription and enrichment amplification of the whole genome product of CVA6 subtype.
[0007] To achieve the above objectives, the present technical solution provides a whole genome capture primer combination for Coxsackie A6, including:
[0008] A first primer pool, wherein the first primer pool includes a first primer pair having a sequence such as SEQ ID NO.1-SEQ ID NO.3, a third primer pair having a sequence such as SEQ ID NO.6-SEQ ID NO.7, a fifth primer pair having a sequence such as SEQ ID NO.10-SEQ ID NO.11, and a seventh primer pair having a sequence such as SEQ ID NO.14-SEQ ID NO.16;
[0009] A second primer pool, wherein the second primer pool includes a second primer pair having a sequence such as SEQ ID NO.4-SEQ ID NO.5, a fourth primer pair having a sequence such as SEQ ID NO.8-SEQ ID NO.9, and a sixth primer pair having a sequence such as SEQ ID NO.12-SEQ ID NO.13;
[0010] The primer sequences of the primer pairs in the first primer pool and the second primer pool are cross-overlapped in sequence.
[0011] The whole genome capture primer combination of Coxsackie A6 provided in this scheme is designed with 7 pairs of primers according to the shingled principle for the whole genome of the CVA6 subtype. There are overlapping sequences between adjacent primer pairs, thereby avoiding the loss of some fragments in the whole genome during the amplification process. In addition, this scheme divides the adjacent primer pairs with overlapping sequences in the 7 pairs of primers into two primer pools, and uses the two primer pools to perform whole genome segmented amplification of the CVA6 subtype to avoid preferential amplification of relatively short overlapping sequences. Finally, the amplified products are mixed to obtain the CVA6 subtype whole genome product, realizing the reverse transcription and enrichment amplification of the CVA6 subtype whole genome. The final DNA product can be used in various molecular biology experiments, including second-generation and third-generation whole genome sequencing experiments.
[0012] In some embodiments, the first primer pair, the second primer pair, the third primer pair, the fourth primer pair, the fifth primer pair, the sixth primer pair and the seventh primer pair are designed by the shingled PCR principle. The primer sequences of the primer pairs in the first primer pool and the second primer pool of this scheme overlap in sequence, which means that the primer pairs corresponding to the even values in the second primer pool have overlapping sequences with the primer pairs corresponding to the adjacent odd values in the first primer pool.
[0013] Specific:
[0014] The second primer pair in the second primer pool has overlapping sequences with the first primer pair and the third primer pair in the first primer pool;
[0015] The fourth primer pair in the second primer pool has overlapping sequences with the third primer pair and the fifth primer pair in the first primer pool;
[0016] The sixth primer pair in the second primer pool has overlapping sequences with the fifth primer pair and the seventh primer pair in the first primer pool.
[0017] In addition, different primer pairs in the first primer pool do not have overlapping sequences, and different primer pairs in the second primer pool do not have overlapping sequences.
[0018] In some embodiments, the first primer pair includes the left primer shown in SEQ ID NO.1, the left primer of SEQ ID NO.2 and the right primer shown in SEQ ID NO.3, the second primer pair includes the left primer shown in SEQ ID NO.4 and the right primer shown in SEQ ID NO.5, the third primer pair includes the left primer shown in SEQ ID NO.6 and the right primer shown in SEQ ID NO.7, the fourth primer pair includes the left primer shown in SEQ ID NO.8 and the right primer shown in SEQ ID NO.9, the fifth primer pair includes the left primer shown in SEQ ID NO.10 and the right primer shown in SEQ ID NO.11, the sixth primer pair includes the left primer shown in SEQ ID NO.12 and the right primer shown in SEQ ID NO.13, and the seventh primer pair includes the left primer shown in SEQ ID NO.14, the right primer shown in SEQ ID NO.15 and the right primer shown in SEQ ID NO.16. The left primer in the same primer pair will bind to the positive strand of the target DNA and extend from the 5' end to the 3' end, and the corresponding right primer will bind to the negative strand of the target DNA and also extend from the 5' end to the 3' end, thereby achieving specific amplification and replication. The primer sequences of the first primer pool and the second primer pool are shown in Table 1:
[0019] Table 1 Primer sequence list
[0020]
[0021]
[0023] In some embodiments, the amplified fragment of each primer pair in the first primer pool and the second primer pool is 1024-1200 bp, preferably 1200 bp in length, to ensure the efficiency and fidelity of the amplified product; and each primer pair is selected in a highly conservative region.
[0024] Specifically, the first primer pair covers the position of 1-1294bp, the second primer pair covers the position of 1102-2381bp, the third primer pair covers the position of 2159-3330bp, the fourth primer pair covers the position of 3190-4469bp, the fifth primer pair covers the position of 4218-5505bp, the sixth primer pair covers the position of 5369-6572bp, and the seventh primer pair covers the position of 6390-7414bp.
[0025] In some embodiments, a forward primer is added to the first primer pair and a reverse primer is added to the seventh primer pair. The advantage of this is that the beginning and end of the reference genome of many viruses are incomplete, and there are only a few sequences to refer to when designing primers. The primer design is prone to off-target, so an extra primer is added at the beginning and end to ensure the amplification effect.
[0026] In a second aspect, the present solution provides a whole genome capture kit for Coxsackie A6, comprising: the whole genome capture primer combination for Coxsackie A6 mentioned in the first aspect and a reverse transcription reagent.
[0027] This protocol utilizes the highly thermostable and efficient reverse transcriptase mentioned in the Coxsackie A6 whole genome capture kit to consistently and rapidly synthesize cDNA from purified total DNA. The primer combination provided in this protocol can be used to amplify the Coxsackie A6 whole genome. The Coxsackie A6 whole genome obtained in this protocol can be used in various molecular biology experiments, including second-generation and third-generation whole genome sequencing experiments.
[0028] In some embodiments, the whole genome capture kit for Coxsackie A6 provided in this solution can be used to amplify the whole genome product of Coxsackie A6, and the whole genome product of Coxsackie A6 can be used in second-generation and third-generation whole genome sequencing experiments.
[0029] In some embodiments, the whole genome capture kit for Coxsackie A6 provided in this embodiment is used to capture the whole genome product of Coxsackie A6 from nucleic acid samples extracted from nasopharyngeal swabs, anal swabs, and herpes fluid environments.
[0030] In a third aspect, the present invention provides a non-diagnostic method for whole genome capture of Coxsackie A6, comprising:
[0031] Obtaining nucleic acid samples to be tested;
[0032] Reverse transcription is performed on the nucleic acid sample to be tested using a reverse transcription reagent to obtain a cDNA chain;
[0033] Performing multiple PCR amplification on the cDNA chain using the first primer pool to obtain a first amplification product;
[0034] Performing multiplex PCR amplification on the cDNA strand using a second primer pool to obtain a second amplification product;
[0035] The first amplification product and the second amplification product were combined to obtain the complete genome of Coxsackie A6.
[0036] In the step of "using reverse transcriptase to reverse transcribe the nucleic acid sample to be tested to obtain a cDNA chain", reverse transcription is performed according to the reaction procedure shown in Table 2 below to obtain a cDNA chain.
[0037] Table 2 Reverse transcription program
[0038] step temperature time cycle 1 25℃ 10 minutes 1 2 50℃ 10 minutes 1 3 85℃ 5 minutes 1 4 4℃ Keep .
[0039] In some embodiments, RT Super Mix is selected as the reverse transcription reagent. RT Super Mix is a premixed reagent for reverse transcription reaction, which usually contains all necessary components for an efficient reverse transcription process, including reverse transcriptase, random primers, dNTPs, buffer, RNase inhibitor and other auxiliary factors.
[0040] In the step of "performing multiplex PCR amplification on the cDNA chain using the first primer pool to obtain the first amplification product, and performing multiplex PCR amplification on the cDNA chain using the second primer pool to obtain the second amplification product", the conditions for the multiplex PCR amplification are the same, namely, cycle 1: 30 seconds at 98°C; cycle 2: 15 seconds at 98°C and 5 minutes at 63°C; cycle 3: 15 seconds at 98°C and 4 minutes at 65°C; cycle 4: 2 minutes at 72°C; cycle 5: maintain at 4°C, wherein cycle 2 is performed for 5 cycles and cycle 3 is performed for 25 cycles.
[0041] The PCR program table for multiplex PCR amplification provided by this protocol is shown in Table 3:
[0042] Table 3 PCR program table
[0043]
[0044]
[0046] In some embodiments, the whole genome of Coxsackie A6 is sequenced, and a second-generation sequencing platform or a third-generation sequencing platform is selected.
[0047] Compared with the prior art, this technical solution has the following characteristics and beneficial effects:
[0048] This scheme designed 7 pairs of primers with overlapping sequence parts through the principle of shingled PCR, and divided the 7 pairs of primers into two primer pools for segmented amplification. The length of each amplified fragment was about 1200bp, ensuring the efficiency and fidelity of the amplified products. Finally, the amplified products were mixed to obtain the CVA6 subtype full genome product. Specifically, this scheme uses 7 pairs of overlapping primers designed by shingled PCR to ensure that the full-length sequence of the CVA6 subtype genome can be amplified, so as to accurately type all CVA6 variants. Degenerate base coverage is designed for different variants to ensure that most CVA6 strains can be successfully amplified. In addition, the 7 pairs of primers designed in this scheme have the advantages of suitable amplification length and strong stability compared with the CVA6 subtype genome. Each pair of primers is selected in the most conservative region, and a forward primer is added to the first pair of primers, and a reverse primer is added to the last pair of primers to ensure the amplification sensitivity and efficiency of different variant samples; at the same time, considering that amplifying the 7 pairs of primers together may result in the primers amplifying relatively short overlapping regions, this scheme divides the 7 pairs of primers into two primer pools for segmented amplification.
[0049] The whole genome capture primer combination, kit and application of Coxsackie A6 provided in this scheme have good compatibility: can achieve whole genome amplification of all variants and typing of CVA6; high coverage: the amplified sequence can cover more than 99% of the complete sequence of CVA6, with uniform coverage; good adaptability. BRIEF DESCRIPTION OF THE DRAWINGS
[0050] Figure 1 It is a schematic diagram of the primer design principles for the first primer pair to the seventh primer pair provided in this scheme.
[0051] Figure 2 This is the assembly analysis result of the sequencing data of Example 1.
[0052] Figure 3 This is the assembly analysis result of the sequencing data of Example 2. DETAILED DESCRIPTION
[0053] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.
[0054] It is to be understood that the term "one" should be understood as "at least one" or "one or more", that is, in one embodiment, the number of an element may be one, while in another embodiment, the number of the element may be multiple, and the term "one" should not be understood as a limitation on the quantity.
[0055] Embodiment 1
[0056] 1. Sample source: 1 CVA6 clinical sample from a disease control center;
[0057] 2. RNA reverse transcription:
[0058] Place 3ul RT super mix and 12ul negative control group into a 15ul mixture in a PCR tube, flick to mix the components in the PCR tube and centrifuge briefly, as shown in Table 4 below:
[0059] Table 4 Reverse transcription program
[0060] step temperature time cycle 1 25℃ 10 minutes 1 2 50℃ 10 minutes 1 3 85℃ 5 minutes 1 4 4℃ Keep .
[0061] 3. Whole genome amplification:
[0062] According to the amplification system shown in Table 5 below, the reaction systems are configured for the first primer pool and the second primer pool respectively:
[0063] Table 5 Amplification system
[0064]
[0065] The reaction system obtained by configuring each primer pool and the reaction system obtained by configuring the second primer pool are respectively subjected to multiple PCR to obtain the first amplification product and the second amplification product, wherein the PCR program table of the multiple PCR program is shown in Table 6 below:
[0066] Table 6 PCR program table
[0067]
[0069] The first amplification product and the second amplification product of the same sample were combined into a 1.5 mL centrifuge tube to obtain the whole genome amplified nucleic acid of CVA6.
[0070] 4. Sequencing:
[0071] The amplified product was purified with 1 volume of magnetic beads. After purification, samples with qubit quantification above 10 ng / ul can be directly used for subsequent library construction experiments. BK-AUX024 and SQK-LSK110 were used for nanopore sequencing library construction and sequencing. The data were assembled and analyzed using assembly software. The analysis results are shown in the figure. Figure 2 As shown, the sequencing data covered 100% of the genome.
[0072] Embodiment 2
[0073] 1. Sample source: inactivated CVA6 isolated and cultured by a disease control center;
[0074] CVA6 RNA was extracted using QIAamp Viral RNA Mini Kit (Cat. No. 52904) produced by Qiagen.
[0075] Reverse transcription, whole genome amplification and sequencing were performed in the same manner as in Example 1 to obtain the analysis results as shown in FIG. Figure 3 As shown, it can be seen that the sequencing data covers 100% of the genome.
[0076] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.
Claims
1. A whole genome capture primer combination for Coxsackie A6, characterized in that: include: A first primer pool, wherein the first primer pool includes a first primer pair having a sequence such as SEQ ID NO.1-SEQ ID NO.3, a third primer pair having a sequence such as SEQ ID NO.6-SEQ ID NO.7, a fifth primer pair having a sequence such as SEQ ID NO.10-SEQ ID NO.11, and a seventh primer pair having a sequence such as SEQ ID NO.14-SEQ ID NO.16; A second primer pool, wherein the second primer pool includes a second primer pair having a sequence such as SEQ ID NO.4-SEQ ID NO.5, a fourth primer pair having a sequence such as SEQ ID NO.8-SEQ ID NO.9, and a sixth primer pair having a sequence such as SEQ ID NO.12-SEQ ID NO.13; The primer sequences of the primer pairs in the first primer pool and the second primer pool are cross-overlapped in sequence.
2. The whole genome capture primer combination of Coxsackie A6 according to claim 1, characterized in that: The primer pairs corresponding to the even values in the second primer pool have overlapping sequences with the primer pairs corresponding to the adjacent odd values in the first primer pool.
3. The whole genome capture primer combination of Coxsackie A6 according to claim 1, characterized in that: The second primer pair in the second primer pool has overlapping sequences with the first primer pair and the third primer pair in the first primer pool, respectively; the fourth primer pair in the second primer pool has overlapping sequences with the third primer pair and the fifth primer pair in the first primer pool, respectively; and the sixth primer pair in the second primer pool has overlapping sequences with the fifth primer pair and the seventh primer pair in the first primer pool, respectively.
4. The whole genome capture primer combination of Coxsackie A6 according to claim 1, characterized in that: Different primer pairs in the first primer pool do not have overlapping sequences, and different primer pairs in the second primer pool do not have overlapping sequences.
5. The whole genome capture primer combination of Coxsackie A6 according to claim 1, characterized in that: The amplified fragment of each primer pair in the first primer pool and the second primer pool is 1024-1200 bp.
6. A whole genome capture kit for Coxsackie A6, characterized in that: include: The whole genome capture primer combination and reverse transcription reagent of Coxsackie A6 according to any one of claims 1 to 5.
7. The whole genome capture kit for Coxsackie A6 according to claim 6, characterized in that: Used to amplify the whole genome product of Coxsackie A6.
8. The whole genome capture kit for Coxsackie A6 according to claim 6, characterized in that: It is designed to capture the whole genome product of Coxsackie A6 in nucleic acid samples extracted from nasopharyngeal swabs, anal swabs, and herpes fluid environments.
9. A non-diagnostic method for whole genome capture of Coxsackie A6, characterized in that include: Obtaining nucleic acid samples to be tested; Reverse transcription is performed on the nucleic acid sample to be tested using a reverse transcription reagent to obtain a cDNA chain; Performing multiple PCR amplification on the cDNA chain using the first primer pool to obtain a first amplification product; Performing multiplex PCR amplification on the cDNA strand using a second primer pool to obtain a second amplification product; The first amplification product and the second amplification product were combined to obtain the complete genome of Coxsackie A6; The first primer pool includes a first primer pair with a sequence such as SEQ ID NO.1-SEQ ID NO.3, a third primer pair with a sequence such as SEQ ID NO.6-SEQ ID NO.7, a fifth primer pair with a sequence such as SEQ ID NO.10-SEQ ID NO.11, and a seventh primer pair with a sequence such as SEQ ID NO.14-SEQ ID NO.16; The second primer pool includes a second primer pair with a sequence such as SEQ ID NO.4-SEQ ID NO.5, a fourth primer pair with a sequence such as SEQ ID NO.8-SEQ ID NO.9, and a sixth primer pair with a sequence such as SEQ ID NO.12-SEQ ID NO.13; The primer sequences of the primer pairs in the first primer pool and the second primer pool are cross-overlapped in sequence.
10. The non-diagnostic method for whole genome capture of Coxsackie A6 according to claim 9, characterized in that The conditions for multiplex PCR amplification were the same, and the conditions for multiplex PCR amplification were as follows: cycle 1: 98°C for 30 seconds; cycle 2: 98°C for 15 seconds and 63°C for 5 minutes; cycle 3: 98°C for 15 seconds and 65°C for 4 minutes; cycle 4: 72°C for 2 minutes; cycle 5: hold at 4°C, wherein cycle 2 was performed for 5 cycles and cycle 3 was performed for 25 cycles.