CN108342509B - Method for enriching vertebrate viral nucleic acids - Google Patents

Method for enriching vertebrate viral nucleic acids Download PDF

Info

Publication number
CN108342509B
CN108342509B CN201810176857.7A CN201810176857A CN108342509B CN 108342509 B CN108342509 B CN 108342509B CN 201810176857 A CN201810176857 A CN 201810176857A CN 108342509 B CN108342509 B CN 108342509B
Authority
CN
China
Prior art keywords
virus
nucleic acid
enriching
tag
double
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201810176857.7A
Other languages
Chinese (zh)
Other versions
CN108342509A (en
Inventor
陈路
姜君
王华贵
邹弈君
张华宇
贾冰清
刘利成
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Macro & Micro Test Bio Tech Co ltd
Original Assignee
Beijing Macro & Micro Test Bio Tech Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Macro & Micro Test Bio Tech Co ltd filed Critical Beijing Macro & Micro Test Bio Tech Co ltd
Publication of CN108342509A publication Critical patent/CN108342509A/en
Application granted granted Critical
Publication of CN108342509B publication Critical patent/CN108342509B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma

Abstract

The invention provides a method for enriching vertebrate virus nucleic acid, which comprises the steps of carrying out conservative amino acid analysis according to a vertebrate virus phylogenetic gene protein to obtain a homologous amino acid sequence group, then obtaining a corresponding merged nucleic acid short sequence group, and specifically enriching virus nucleic acid in a sample by a method of amplifying and enriching virus sequences. The method can specifically enrich the virus DNA/RNA in the sample, achieve the effect of specifically enriching the virus DNA/RNA in the sample, effectively improve the proportion of the virus DNA/RNA and the host DNA/RNA in the infected sample, improve the proportion of virus sequences in subsequent high-throughput sequencing and improve the sensitivity of detecting the virus.

Description

Method for enriching vertebrate viral nucleic acids
Technical Field
The invention relates to the field of virus detection, in particular to a method for enriching vertebrate virus nucleic acid.
Background
In a virus infection sample, a sample to be detected usually contains a large amount of host genome or transcriptome components besides virus nucleic acid itself, and the components greatly interfere with effective detection of the virus nucleic acid, and the ratio of the virus nucleic acid is usually not high, so that the virus nucleic acid is more difficult to detect, for example, in fluorescence PCR, a low copy number total DNA/RNA sample can generate a missing detection condition; in both macrotranscriptome and metagenomic sequencing, the data volume of the host in some samples can account for more than 99% of the total data volume, while the data volume of viral DNA/RNA molecules is less than 1%. The virus content is too low, the screening of related virus DNA/RNA molecules from macro transcriptome or macro genome sequencing big data is very difficult, huge data volume is needed for supporting, and the sequencing cost and the analysis difficulty are greatly improved. In the face of the situation, the targeted enrichment of vertebrate virus DNA/RNA molecules which we want to study can effectively increase the nucleic acid components and data volume of the virus and improve the success rate and efficiency of subsequent detection.
Currently, the enrichment technique for viral DNA/RNA samples in infected samples is mainly the removal of host genome or transcriptome components. For example, the method of using a human ribosome sequence probe hybridization method to remove the human ribosome transcription component in an infected sample has a low enrichment effect which can only reach an effect of less than one time, and low-copy virus nucleic acid can be lost after enrichment due to the presence of non-specific hybridization. Secondly, the non-target enrichment of the nucleic acid of the micro sample is carried out by utilizing a random amplification method. For example, Sequence Independent Single Primer Amplification (SISPA), which randomly amplifies a micro-infection sample including a host genome and a virus genome, is not effective in enriching a virus genome (virus DNA/RNA). Therefore, there is a need for a virus DNA/RNA enrichment method, which can supplement, optimize and improve the existing virus enrichment methods in terms of range, so as to efficiently and economically increase the ratio of viral genomes in an infected sample and improve the efficiency of subsequent detection, such as nucleic acid amplification, transcriptome sequencing, metagenome sequencing, and the like.
Disclosure of Invention
The invention aims to provide a method for enriching vertebrate virus DNA/RNA, which has a specific enrichment effect on virus DNA/RNA in a sample, can effectively improve the content of virus DNA/RNA in an infected sample, and can improve the virus nucleic acid data ratio in subsequent high-throughput sequencing. This method is called Sequence Dependent Single Primer Amplification (SDSPA).
In one embodiment, a method for enriching a vertebrate viral nucleic acid is provided, the method comprising performing a conservative amino acid analysis on the vertebrate viral phylogenetic gene proteins to obtain a set of homologous amino acid sequences, and then obtaining a set of corresponding short sequences of degenerate nucleic acids, and specifically enriching the viral nucleic acid in a sample by amplification of the enriched viral sequences.
In one embodiment, the number of amino acids of the group of homologous amino acid sequences is from 2 to 10, preferably from 5 to 7.
In one embodiment, when a DNA virus, the viral phylogenetic gene protein is selected from the group consisting of DNA-dependent RNA polymerase (DDRP), nucleoprotein and capsid protein genes; and when an RNA virus, the selected virus phylogenetic gene protein is an RNA-dependent RNA polymerase (RDRP), Nucleoprotein (NP) or Capsid Protein (CP).
In one embodiment, the amplification-enriched viral sequence method is an asymmetric amplification-enriched viral sequence method.
In one embodiment, the method for enriching viral sequences by asymmetric amplification comprises the following steps:
designing a degenerate primer group of the degenerate nucleic acid short sequence group;
b, reverse transcription is carried out on the extracted virus total RNA by utilizing a degenerate primer group with a first label sequence to obtain cDNA with the first label or single-strand extension reaction is carried out on virus dsDNA/ssDNA to obtain ssDNA with the first label;
c, performing double-strand synthesis on the cDNA with the tag or the ssDNA with the tag by using a random primer with a second tag sequence to obtain double-end tag double-strand DNA;
purifying the double-stranded DNA containing the double-ended tag;
and e, performing PCR amplification on the double-end-tagged double-stranded DNA by using the double-end tag in the double-end-tagged double-stranded DNA in the step d, and enriching the virus nucleic acid in the sample.
In one embodiment, the first tag and the second tag are nucleic acid sequences having a length of 18 to 22 bases, a GC content percentage of 40 to 60%, a Tm of 58 + -5 deg.C, 58 + -3 deg.C or 58 + -1 deg.C; the first label and the second label do not cross the degenerate primer group after being subjected to Blast comparison.
In one embodiment, the first and second tags are the same. In one embodiment, the first and second labels are both GCCGGAGCTCTGCAGAATTC.
In one embodiment, the step d is performed by magnetic bead purification to remove the residual random primers.
In one embodiment, the sample is plasma, cerebrospinal fluid, pleural effusion, sputum, urine, nasal wash or anal swab.
In one embodiment, the genes phylogenetically evolved by different ones of the vertebrate viruses, the conservative amino acid analysis to obtain sets of homologous amino acid sequences and the corresponding sets of short degenerate nucleic acid sequences obtained are as follows:
Figure BDA0001587562640000031
Figure BDA0001587562640000041
Figure BDA0001587562640000051
Figure BDA0001587562640000061
drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic diagram of the method for enriching viral sequences by asymmetric amplification according to the present invention.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below with reference to the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Experimental procedures in which specific conditions are not noted in the following examples are generally performed under conventional conditions, such as those described in molecular cloning instructions (third edition), Joseph Sambrook et al, or as suggested by the manufacturer. The various chemicals used in the examples are commercially available products.
The first embodiment is as follows: the enrichment effect of the method (SDSPA) on clinical RNA virus samples is compared with the enrichment effect of SISPA without enrichment.
1. As shown in FIG. 1, 4 clinical samples of RNA were extracted from human serum (Flaviviridae), serum (Flaviviridae) from patients infected with dengue-II virus, anal swab (Caliciviridae) from patients infected with norovirus, and anal swab (Picaviridae) from patients infected with Enterovirus EV71 using RNeasy Mini Kit (Qiagen) as indicated in FIG. 1. Aiming at the four pathogens, a homologous amino acid sequence group GDDCV on an RNA polymerase section which is dependent on the virus phylogenetic gene protein RNA is found, a corresponding degenerate nucleic acid short sequence NACRCARTCRTCNCC is found, and a synthesized tagged sequence TAG-13 is GCCGGAGCTCTGCAGAATTCNACRCARTCRTCNCC.
2. Synthesis of SuperScript Using First-Strand cDNATMII RT the total RNA extracted from 4 infected patient samples was subjected to reverse transcription, the reagents were thawed on ice, the reagents were added according to the system given below and briefly centrifuged.
Figure BDA0001587562640000071
5min at 65 ℃; immediately placed on ice for 3min and the following reagents were added to the reaction tube:
Figure BDA0001587562640000072
mix by gentle inversion, centrifuge briefly, and centrifuge at 42 ℃ for 2 min.
Add 1. mu.L SuperScriptTMII RT (200units), mix by gentle inversion, centrifuge briefly, 50min at 42 ℃ and 15min at 72 ℃. The cDNA product containing tag GCCGGAGCTCTGCAGAATTC was obtained.
3. Second strand synthesis of the reverse-transcribed single-stranded cDNA was performed with phi29DNA polymerase (NEB). The reaction system is as follows:
Figure BDA0001587562640000073
Figure BDA0001587562640000081
at 95 ℃ for 3min, and quenched in ice bath for 5 min.
Figure BDA0001587562640000082
2h at 30 ℃ and 10min at 65 ℃. A dsDNA product containing double-ended GCCGGAGCTCTGCAGAATT C was obtained. TAG-N8 in the table is GCCGGAGCTCTGCAGAATTCNNN NNNNN; the sequence of the 8 first halves of N is the label, and is consistent with the label of the first step.
4. Magnetic bead purification, adopting 1.0-1.2 times magnetic bead to carry out primary fragment screening, obtaining pure dsDNA products with double-end labels, which do not contain residual primers, primer dimers generated by primer self-connection, residual buffer ions of various reaction systems and the like, so as to reduce non-specific amplification in the second round of amplification.
5. The purified product was subjected to tag enrichment amplification with the high fidelity amplification enzyme Kapa Biosystems HiFi HS (kk2600) polymerase in the following reaction scheme:
the PCR reaction was carried out under the following conditions: the template DNA was denatured and kept at 95 ℃ for 5 min. PCR reaction
Figure BDA0001587562640000083
Circulation conditions are as follows:
the following 20 cycles were performed:
step 1: at 95 ℃ for 20 s;
step 2: at 54 ℃ for 20 s;
and 3, step 3: 35s at 72 ℃;
a large amount of dsDNA product of double-ended tag GCCGGAGCTCTGCAGAATTC was obtained.
6. Purification was performed again using 1.0-1.2 Xmagnetic beads to obtain a pure dsDNA product of double-ended tag GCCGGAGCTCTGCAGAATTC free of dNTPs, primers, primer dimers, salt ions and other impurities.
7. The specific fluorescence PCR of the pathogen and the fluorescence detection system of the human internal reference (. beta. -ACTIN) were used to detect the products of 6 and the corresponding products of SISPA technology, respectively, and the Ct values are shown in the following table:
Figure BDA0001587562640000091
8. standard DNA Library construction is carried out on the product of 6 and the corresponding product of SISPA by adopting a VAHTS Universal DNA Library Prep Kit for illumana V2 Kit, sequencing is carried out by adopting a PE125 sequencing mode by utilizing an illumana Nextseq500 sequencer, the sequencing quantity of each Library is about 20M reads (about 2.5G data quantity), and the sequencing reads number is shown in the following table:
Figure BDA0001587562640000092
the results show that:
as can be seen from the results in the above table, the combined enrichment product prepared by the method aiming at the virus infection sample has a good enrichment effect on the virus RNA to be detected, and when the same data volume is tested (20 Mreads, 2.5G data volume), the ratio of the number of the virus sequences obtained by sequencing the sample after enrichment is greatly improved compared with the ratio of the virus content without enrichment treatment, which indicates that the technology can effectively enrich the virus components. Meanwhile, when the sample is a trace sample and the direct sequencing of the nucleic acid extracted from the sample cannot meet the sequencing requirement, the nucleic acid is generally amplified and enriched by adopting a SISPA method, as can be seen from the above table, although the total amount of the nucleic acid can be amplified by the SISPA technology, the virus enrichment effect is basically not obtained, even the virus nucleic acid content ratio is reduced, and the virus amplification and enrichment effect of the method of the invention is obviously improved compared with the virus amplification and enrichment effect of the SISPA on the trace sample.
Example two: the enrichment effect of the method (SDSPA) on the DNA virus infection sample is compared with the enrichment effect of the method without enrichment and adopting SISPA
1. As shown in FIG. 1, DNA of human throat swab (adenovirus) infected with adenovirus type 7, human serum (herpesvirus family) infected with herpes simplex Virus type 1, and allantoic fluid (chordopoxviridae family) infected with fowlpox Virus was extracted using QIAamp MinElute Virus Spin Kit (Qiagen). Aiming at the three pathogens, a merged nucleic acid short sequence NSWRTC NGTRTCNCCRTA corresponding to the homologous amino acid sequence group YGDTDS on the virus phylogenetic gene protein DNA polymerase section is found, and a tagged sequence TAG-1GCCGGAGCTCTGCAGAATT CNSWRTCNGTRTCNCCRTA is synthesized.
2. The 3 pathogen total DNAs were subjected to single primer extension reaction using Bst DNA POLYMERASE (NEB), each reaction reagent was thawed on ice, the corresponding reagents were added according to the following table system, and briefly centrifuged.
Figure BDA0001587562640000101
Mix by gentle inversion, centrifuge briefly, and the reaction procedure is as follows:
step 1: extending for 60min at 63 ℃;
step 2: inactivating enzyme at 80 deg.C for 15 min;
ssDNA products containing tag GCCGGAGCTCTGCAGAATTC were obtained.
3. And (3) purifying by using an ultrathin DNA product purification kit to obtain a pure ssDNA product with the tag GCCGGAGCTCTGCAGAATTC and without buffer ion impurities in a related system.
4. Second strand synthesis of single-stranded ssDNA obtained from the single primer extension reaction was performed with phi29DNA polymerase (NEB). The reaction system is as follows:
Figure BDA0001587562640000102
Figure BDA0001587562640000111
at 95 ℃ for 3min, and quenched in ice bath for 5 min.
Figure BDA0001587562640000112
2h at 30 ℃ and 10min at 65 ℃. dsDNA products containing the double-ended GCCGGAGCTCTGCAGAATTC sequence were obtained.
5. Magnetic bead purification, using 1.0-1.2 × magnetic bead for one fragment screening to reduce non-specific amplification during the second round of amplification. Obtaining pure dsDNA products with double-end labels without residual primers, primer dimers generated by primer self-ligation and residual buffer ions and the like in various reaction systems so as to reduce non-specific amplification in the second round of amplification.
6. The purified product was subjected to tag enrichment amplification with high fidelity amplification enzyme Kapa biosystemmsHiFi HS (kk2600) polymerase in the following reaction scheme:
Figure BDA0001587562640000113
the PCR reaction was carried out under the following conditions: the template DNA was denatured and kept at 95 ℃ for 5 min. PCR cycling conditions:
the following 20 cycles were performed:
step 1: at 95 ℃ for 20 s;
step 2: at 54 ℃ for 20 s;
and 3, step 3: 35s at 72 ℃;
a large amount of dsDNA product of double-ended tag GCCGGAGCTCTGCAGAATTC was obtained.
7. Purification was performed again using 1.0-1.2 Xmagnetic beads to obtain a pure dsDNA product of double-ended tag GCCGGAGCTCTGCAGAATTC free of dNTPs, primers, primer dimers, salt ions and other impurities.
8. The specific fluorescence PCR of the pathogen, the products of 6 and corresponding SISPA technical products were detected respectively by using the fluorescence detection systems of human internal reference (. beta. -ACTIN) and chicken. beta. -ACTIN, and Ct values are shown in the following table:
Figure BDA0001587562640000121
9. standard DNA Library construction was performed on products of 6 and corresponding SISPA products using VAHTS Universal DNA Library Prep Kit for illumana V2 Kit, sequencing was performed using the illumana Nextseq500 sequencer using PE125 sequencing mode, sequencing amount for each Library was about 20M reads (-2.5G data amount), sequencing reads number is shown in the following table:
Figure BDA0001587562640000122
the results show that:
as can be seen from the results in the above table, the facultative enrichment product prepared by the method aiming at the virus infection sample has a good enrichment effect on the virus DNA to be detected, and when the same data volume is tested (20M reads, 2.5G data volume), the ratio of the number of the virus sequences obtained by sequencing the enriched sample is greatly improved compared with the ratio of the virus content without enrichment treatment, which indicates that the SDSPA technology can effectively enrich the virus components. Meanwhile, when the sample is a trace sample and the direct sequencing of the nucleic acid extracted from the sample cannot meet the sequencing requirement, the nucleic acid is generally amplified and enriched by adopting a SISPA method, as can be seen from the table above, although the SISPA technology can amplify the nucleic acid amount, the virus enrichment effect is basically not obtained, even the virus nucleic acid proportion is reduced, and the virus enrichment effect is obviously improved compared with the SISPA when the trace sample is amplified and enriched by adopting the virus enrichment method.
The results of the two examples show that the method provided by the invention can be used for preparing the facultative enrichment product aiming at the virus infection sample, and has a good enrichment effect on the virus DNA/RNA molecules to be detected in the sample. For micro samples, the method has better effect than the SISPA method for enriching the virus nucleic acid. The results show that the method and the technology can effectively enrich the virus nucleic acid (DNA/RNA) in the sample to be detected for subsequent detection and analysis; for micro samples, the method has better capability of enriching the nucleic acid of the virus to be detected than SISPA.
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (7)

1. A method for enriching a vertebrate viral nucleic acid, comprising performing conservative amino acid analysis on the vertebrate viral phylogenetic gene protein to obtain a homologous amino acid sequence group, then obtaining a corresponding merged nucleic acid short sequence group, and specifically enriching the viral nucleic acid in a sample by a method of amplifying and enriching the viral sequence;
the genes evolved by different vertebrate virus systems, conservative amino acid analysis to obtain homologous amino acid sequence groups and the obtained corresponding merged nucleic acid short sequence groups are respectively shown in the following table:
Figure 235775DEST_PATH_IMAGE001
Figure 966926DEST_PATH_IMAGE002
Figure 320547DEST_PATH_IMAGE003
；
the virus sequence amplification and enrichment method is an asymmetric virus sequence amplification and enrichment method;
the method for enriching the virus sequences by asymmetric amplification comprises the following steps:
designing a degenerate primer group of the degenerate nucleic acid short sequence group;
b, reverse transcription is carried out on the extracted virus total RNA by utilizing a degenerate primer group with a first label sequence to obtain cDNA with the first label or single-strand extension reaction is carried out on virus dsDNA/ssDNA to obtain ssDNA with the first label;
c, performing double-strand synthesis on the cDNA with the tag or the ssDNA with the tag by using a random primer with a second tag sequence to obtain double-end tag double-strand DNA;
purifying the double-stranded DNA containing the double-ended tag;
and e, performing PCR amplification on the double-end-tagged double-stranded DNA by using the double-end tag in the double-end-tagged double-stranded DNA purified in the step d, and enriching the virus nucleic acid in the sample.
2. A method for enriching nucleic acid of a vertebrate virus according to claim 1 wherein the protein of the selected virus phylogenetic gene is a DNA-dependent RNA polymerase, nucleoprotein or capsid protein; and when an RNA virus, the selected virus phylogenetic gene protein is an RNA-dependent RNA polymerase, nucleoprotein or capsid protein.
3. The method for enriching vertebrate viral nucleic acid according to claim 1, wherein said first tag and said second tag are nucleic acid sequences having a length of 18 to 22 bases, a percentage of GC content of 40 to 60%, and a Tm value of 58 ± 5 ℃; the first label and the second label do not cross the degenerate primer group after being subjected to Blast comparison.
4. The method for enriching for vertebrate viral nucleic acid according to claim 3, wherein said first tag and said second tag are the same.
5. The method of claim 4, wherein the first tag and the second tag are both GCCGGAGCTCTGCAGAATTC.
6. The method for enriching nucleic acid of vertebrate viruses according to claim 1, wherein the step d comprises removing residual random primers by magnetic bead purification.
7. The method for enriching vertebrate viral nucleic acid according to claim 1, wherein the sample is plasma, cerebrospinal fluid, pleural effusion, sputum, urine, nasal wash or anal swab.
CN201810176857.7A 2018-02-08 2018-03-04 Method for enriching vertebrate viral nucleic acids Active CN108342509B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810130657 2018-02-08
CN2018101306578 2018-02-08

Publications (2)

Publication Number Publication Date
CN108342509A CN108342509A (en) 2018-07-31
CN108342509B true CN108342509B (en) 2021-07-09

Family

ID=62960010

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810176857.7A Active CN108342509B (en) 2018-02-08 2018-03-04 Method for enriching vertebrate viral nucleic acids

Country Status (1)

Country Link
CN (1) CN108342509B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110093455B (en) * 2019-04-27 2020-03-17 中国医学科学院病原生物学研究所 Respiratory virus detection method

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999053097A2 (en) * 1998-04-15 1999-10-21 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Centers For Disease Control And Prevention Method for developing degenerate pcr primers
US8032310B2 (en) * 2004-07-02 2011-10-04 The United States Of America As Represented By The Secretary Of The Navy Computer-implemented method, computer readable storage medium, and apparatus for identification of a biological sequence
WO2011022503A1 (en) * 2009-08-19 2011-02-24 Blood Systems, Inc. New astrovirus species
CN102352366B (en) * 2011-09-28 2013-08-14 浙江省淡水水产研究所 Macrobrachium rosenbergii bicistronic messenger ribonucleic acid (mRNA) viral genome complete sequence and application thereof
CN102643929A (en) * 2012-04-05 2012-08-22 中华人民共和国大榭出入境检验检疫局 Consensus-degenerate hybridoligonucleotide primer (CODEHOP) reverse transcription-polymerase chain reaction (RT-PCR) reagent and method for detecting lyssavirus viruses
CN105648112A (en) * 2016-02-21 2016-06-08 杨亚伟 General sequencing method for DHAV (duck hepatitis A virus) genome sequence

Also Published As

Publication number Publication date
CN108342509A (en) 2018-07-31

Similar Documents

Publication Publication Date Title
CN110093455B (en) Respiratory virus detection method
Moldovan et al. Methodological challenges in utilizing mi RNA s as circulating biomarkers
Hall et al. Evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery
Datta et al. Next-generation sequencing in clinical virology: Discovery of new viruses
Rosseel et al. Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples
He et al. Rapid and highly sensitive one-tube colorimetric RT-LAMP assay for visual detection of SARS-CoV-2 RNA
Goya et al. An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates
Ke et al. Colorimetric nucleic acid testing assay for RNA virus detection based on circle-to-circle amplification of padlock probes
CN113337639B (en) Method for detecting COVID-19 based on mNGS and application thereof
EP3350351B1 (en) Virome capture sequencing platform, methods of designing and constructing and methods of using
Van Nostrand et al. Variation in single-nucleotide sensitivity of eCLIP derived from reverse transcription conditions
Alfson et al. A new approach to determining whole viral genomic sequences including termini using a single deep sequencing run
Odiwuor et al. A point-of-care SARS-CoV-2 test based on reverse transcription loop-mediated isothermal amplification without RNA extraction with diagnostic performance same as RT-PCR
CN112813140A (en) Nucleic acid releasing agent and nucleic acid releasing method thereof
CN108342509B (en) Method for enriching vertebrate viral nucleic acids
CN105950785B (en) Triple fluorescence RT-PCR detection kit, primer and probe for avian influenza virus, newcastle disease virus and infectious bronchitis virus
CN103173568B (en) Primer group, kit and method for rapidly identifying 1 type dengue fever virus
CN113430303A (en) Rapid identification method for 23 respiratory RNA viruses based on nanopore sequencer
Qian et al. Hepatitis C Virus Infection Cycle-Specific MicroRNA Profiling Reveals Stage-Specific miR-4423-3p Targets RIG-I to Facilitate Infection
WO2023202030A1 (en) Method for constructing high-throughput sequencing library of small rna
Xi et al. A single mutation in the cis-acting replication element identified within the EV-A71 2C-coding region causes defects in virus production in cell culture
Parreira Laboratory methods in molecular epidemiology: viral infections
CN105936944B (en) Triple fluorescent RT-PCR detection kit, primer and probe for CSFV, PRRSV and SIV H1 subtypes
Wu et al. Ultrasensitive detection of serum hepatitis B virus by coupling ultrafiltration DNA extraction with real-time PCR
CN110684862B (en) Microdroplet digital PCR kit for quantitatively detecting hepatitis B virus and detection method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant