CN117646088A - Human metapneumovirus whole genome enrichment kit for sequencing analysis - Google Patents
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Abstract
The invention discloses a human metapneumovirus whole genome enrichment kit for sequencing analysis, and relates to the technical field of biology. The invention provides a primer combination for enriching human metapneumovirus whole genome for sequencing analysis, which comprises 29 primer sequences, wherein the 29 primer sequences are shown as SEQ ID NO. 1-SEQ ID NO. 29. The primer combination adopts shingled arrangement on the sequence, and well covers the whole genome sequence of the metapneumovirus. The primer group provided by the invention captures and builds a library for sequencing aiming at different samples, the sequencing depth is more than or equal to 1000 multiplied by the total genome coverage can still reach more than 99%, and the primer group has very high coverage uniformity.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a human metapneumovirus whole genome enrichment kit for sequencing analysis.
Background
Human metapneumovirus (human metapneumovirus, HMPV) is a common human respiratory pathogen, and particles are polymorphic, spherical, fibrous under electron microscopy. HMPV belongs to the pneumoviridae family, genus metapneumovirus, enveloped single-stranded negative-strand RNA viruses. HMPV contains 8 genes and 9 open reading frames, and the encoded proteins mainly comprise genes such as nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), transcription elongation factor (M2-1), RNA synthesis regulator (M2-2), small hydrophobic surface protein, adhesion protein (G), polymerase (L) subunits and the like. HMPV shares A, B genotypes and four genotypes A1, A2, B1, B2, with the A2 subtype being further divided into the A2a and A2B gene clusters.
In 2001, the first time it was detected by the netherlands scholars from nasopharyngeal aspirate samples of infected children. HMPV is transmitted primarily through the respiratory system, and coughing and sneezing, intimate personal contact and touch of viral contaminants can all contribute to transmission. In general, the incubation period after infection is about 3-5 days, the detection rate is highest in winter and spring, and repeated infection is common. HMPV infection can also cause outbreaks, especially in children with symptoms such as hypoxia, fever, coughing, wheezing.
There are three commonly used detection methods for HMPV at present, including: antigen antibody detection, virus cell culture and reverse transcription polymerase chain reaction (RT-PCR). The virus cell culture technique has higher requirements, longer culture time, difficult recognition of cytopathic effect and lower sensitivity. The antigen detection of HMPV is commonly used in clinic by immunofluorescence, immunochromatography and enzyme immunoassay. However, these detection techniques can only identify the pathogen, but cannot analyze the whole genome sequence of the pathogen, and cannot further track and trace the epidemic variation of the virus.
Chinese patent CN101446592a discloses an indirect immunofluorescence detection kit for human metapneumovirus, said kit comprising reagent a: murine anti-human metapneumovirus monoclonal antibody: 1ml of a 1% BSA solution with a volume percentage of 1:50-20000; reagent B: 100ml of phosphate buffer containing Tween 20; reagent C: FITC-labeled goat anti-mouse IgG antibody: 1ml of solution with the volume percentage of the reagent B being 1:10-1000; reagent D: 1ml of Evansi blue dye solution with the concentration of 0.01 percent. However, the sensitivity and specificity of the kit provided by the invention are worse than those of RT-PCR. The RT-PCR technique is the most sensitive and effective method for detecting HMPV infection, and has high sensitivity and short reaction time.
The invention develops a whole set of primers which can cover the whole genome sequence of the human metapneumovirus for downstream library-building sequencing analysis, thereby being beneficial to better monitoring the flow trend and variation condition of the human metapneumovirus in China and preventing sudden epidemic.
Disclosure of Invention
The invention aims to provide a human metapneumovirus whole genome enrichment kit for sequencing analysis, and the primer combination of the kit well covers the whole genome sequence of the metapneumovirus and has high coverage uniformity.
In order to achieve the above object, the present invention has the following technical scheme:
in one aspect, the invention provides a primer combination for enriching human metapneumovirus whole genome for sequencing analysis, wherein the primer combination comprises 29 primer sequences, and the 29 primer sequences are shown as SEQ ID NO. 1-SEQ ID NO. 29.
SEQ ID NO. 1 is: AAAATGTCTCTTCAAGGGATTCACC.
SEQ ID NO. 2 is: TGACTTGTCCCASTTTTTTAATTACTC.
SEQ ID NO. 3 is: CACAGATGAAGAAAAAGARGCTGC.
SEQ ID NO. 4 is: CTTTTTCTACYAGATCAACTTGAACAGC.
SEQ ID NO. 5 is: GGGACAAGTMAARATGGAGTCCTA.
SEQ ID NO. 6 is: TCATGTAGYACTATAACTGARGGATA.
SEQ ID NO. 7 is: GGGACAARTAAAAATGTCTTGGAAAGT.
SEQ ID NO. 8 is: CCATRCTGATAGGRTGYCTTCCTGT.
SEQ ID NO. 9 is: TCAARRGARTGCAACATCAACATATC.
SEQ ID NO. 10 is: CAGGAGTTTTGCTCATCTCCAT.
SEQ ID NO. 11 is: GCTGATGGYYTRTCAATAATATCAGG.
SEQ ID NO. 12 is: TTCCATTCRATTGTGTATATACAATGGCA.
SEQ ID NO. 13 is: GCTGGAAAATAAGCAGAAATCAATG.
SEQ ID NO. 14 is: GGATCCATTGTTATTTRTCYCTT.
SEQ ID NO. 15 is: ATGCARCATGAAATAATGAARAAT.
SEQ ID NO. 16 is: MAAGRGAYAAATAACAATGGATCC.
SEQ ID NO. 17 is: TTCTGAATATATARTATAATTCTGCAGCA.
SEQ ID NO. 18 is: CAACTGTTAACATGGAAAGATGTGATG.
SEQ ID NO. 19 is: AGAAGAAAGTTCYGATTTGATTTCCAT.
SEQ ID NO. 20 is: TGATYTAGATCTYCARAGAATWATGGAAAT.
SEQ ID NO. 21 is: CTGCTGTTCTATTKATATCACTTGTTAC.
SEQ ID NO. 22 is: ACAACACTAATGAGAGAYCCTCAGG.
SEQ ID NO. 23 is: CACTCATTATRCTAATTCCRCAGCT.
SEQ ID NO. 24 is: CWGTTCCAGCTTATAGRACAACAAATTA.
SEQ ID NO. 25 is: CGTGTRAGAGCATGTGCCAT.
SEQ ID NO. 26 is: AGAATGTTCAGCAARGTYATGTTTGA.
SEQ ID NO. 27 is: CACTGCTATTYTCATYTTRGAATTTTGTAT.
SEQ ID NO. 28 is: GATGCTTTATTRATAACWTTRTGTGATGCAGA.
SEQ ID NO. 29 is: ATCTCTGCATTYCCYAGGTTATCTAT.
In yet another aspect, the invention provides the use of the primer combination described above for detecting human metapneumovirus.
In particular, the above-mentioned applications are for diagnosis, genotyping and sample tracing of human metapneumoviruses.
In yet another aspect, the invention provides a kit comprising the primer combination described above.
Preferably, the reaction system of the kit comprises a reverse transcription reaction system and an amplification system.
Further preferably, the reverse transcription reaction system comprises: reverse transcriptase (Enzyme Mix), random hexamers, reverse transcription reaction solution (RT Mix).
Still further preferably, the reverse transcription reaction system includes:
| component (A) | Addition (ul) |
| 10X RT Mix | 2 |
| Enzyme Mix | 2 |
| Random hexamers | 1 |
| RNA | 10 |
| H 2 O | 5 |
Further preferably, the amplification system comprises: high fidelity enzyme, taq enzyme, reaction amplification solution, dNTPs (all contained in 2 XMasterMix).
Still further preferably, the amplification system comprises:
still further, the final concentration of the primer combination in the amplification system is 300nM.
Preferably, the reverse transcription procedure of the kit is: program 1: cycling for 1 time at 25 ℃ for 5 min; program 2: cycling for 1 time at 55 ℃ for 10 min; program 3: and (3) cycling for 1 time at 95 ℃ for 1 min.
Preferably, the amplification procedure of the kit is: program 1: cycling for 1 time at 95 ℃ for 30 sec; program 2: cycling for 30 times at 95 ℃,15sec,60 ℃ for 3 min; maintained at 4 ℃.
Preferably, the kit is for sequencing analysis.
Preferably, the detection sample of the kit is one or more of nasopharyngeal swab, pharyngeal swab, saliva, sputum and alveolar lavage.
Preferably, the detection method of the kit comprises the following steps:
(1) Extracting RNA of a sample to be detected, and carrying out reverse transcription on the RNA into cDNA;
(2) Carrying out PCR amplification on the cDNA of the reverse transcription product;
(3) Library construction was performed and the PCR amplification products were sequenced.
Preferably, the sequence of the primer used in the PCR amplification in the step (2) is shown in SEQ ID NOS.1-29.
Preferably, the library construction step in step (3) is as follows:
(1) Combining and supplementing the amplified products of the two primer groups 1 and 2 to a total volume of 100 mu L, and purifying with 180 mu L of purification magnetic beads;
(2) Adding the purified product of the previous round into ATM and TD components respectively for fragmentation;
(3) The fragmented sequences are amplified by Index to complete library construction;
(4) Library quality inspection was performed.
In yet another aspect, the invention provides the use of the above-described kit for detecting human metapneumovirus.
The beneficial effects of the invention are as follows:
the invention provides a primer combination and a kit for enriching the whole genome of a human metapneumovirus for sequencing, wherein the kit can enrich all subtypes, and the whole genome coverage rate reaches more than 99% in the subsequent connection library-building sequencing process.
Drawings
FIG. 1 is a whole genome coverage map of a human metapneumovirus sample S1.
FIG. 2 is a whole genome coverage map of a human metapneumovirus sample S2.
FIG. 3 is a whole genome coverage map of a human metapneumovirus sample S3.
FIG. 4 is a whole genome coverage map of human metapneumovirus sample S4.
FIG. 5 is a whole genome coverage map of a comparative human metapneumovirus sample S1.
FIG. 6 is a full genome coverage map of a comparative human metapneumovirus sample S2.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purpose and the effect of the present invention easy to understand, the present invention will be further elucidated with reference to the specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all of them. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. In the following examples, unless otherwise specified, the methods of operation used were conventional, the equipment used was conventional, and the materials used in the examples were the same.
Example 1
Primers for detection of human metapneumovirus are shown in table 1 below.
TABLE 1 primer sequences
The nucleotides of the degenerate positions in the above table are denoted r=a/G, y=c/T, m=a/C, k=g/T, s=c/G, w=a/T.
The detection of coverage rate includes the following steps:
whole gene capture procedure:
reverse transcription reaction:
the system comprises: using Norvigilance Co LtdIII 1st Strand cDNA Synthesis Kit,R312, the reaction system is shown in Table 2 below.
Table 2.
The reaction procedure: program 1: cycling for 1 time at 25 ℃ for 5 min; program 2: cycling for 1 time at 55 ℃ for 10 min; program 3: and (3) cycling for 1 time at 95 ℃ for 1 min.
Multiplex amplification reaction:
reagent: NEB company Q5 2x Master mix (M0494L), the two primer sets were amplified separately, and the reaction system was as shown in Table 3.
Table 3.
| Reagent name | Addition amount of |
| Q5 2x MasterMix | 12.5μL |
| Primer group 1/2%10uM) | 4μL |
| Nuclease-free water | 6μL |
| Reverse transcription product | 2.5μL |
| Totalizing | 25μL |
The reaction procedure: program 1: cycling for 1 time at 95 ℃ for 30 sec; program 2: cycling for 30 times at 95 ℃,15sec,60 ℃ for 3 min; maintained at 4 ℃.
The library construction was carried out using the method of Nextera XT Library Preparation Kit (product number: FC-131-1024) from Illumina, inc. and the specific procedure is as follows:
(1) Combining and supplementing the amplified products of the two primer groups 1 and 2 to a total volume of 100 mu L, and purifying with 180 mu L of purification magnetic beads;
(2) Adding the purified product of the previous round into ATM and TD components respectively for fragmentation;
(3) The fragmented sequences are amplified by Index to complete library construction;
(4) Library quality inspection is performed: by usingThe dsDNA quantification kit (available from ThermoFisher company under the product number Q32854) is used for library quantification, and the yield of the qualified library is more than 50 ng.
The library was sequenced after construction and the on-press data were assembled using commercial bioinformatics assembly software (e.g., qiagen. Clc Genomics Workbench) to analyze whole genome coverage and sequence quality.
S1-S4 sample sources: a throat swab sample from a patient infected with human metapneumovirus. After extraction of the commercial nucleic acid kit, the sample nucleic acid Ct was determined using a human metapneumovirus nucleic acid real-time fluorescent PCR detection kit (A3881, zhuo Chenghui).
Experimental results of coverage:
table 4.
The results illustrate: the whole genome length of the sample S1-4 is 13350bp, the coverage rate of the whole genome can be more than 99% for more than 1000X, and the whole genome coverage rate of the whole genome which is more than or equal to 20% of the average depth reaches more than 90%, which indicates that the whole sequence capturing uniformity of the technical method is good, and the whole genome coverage maps of the human metapneumovirus sample S1-S4 are respectively shown in figures 1-4.
Comparative example 1
Primers for detection of human metapneumovirus are shown in table 5 below.
Table 5.
The comparative primer set comprises two sets of primers, primer set 3 and primer set 4.
The detection method of coverage is the same as in example 1.
Whole gene capture procedure:
reverse transcription reaction:
the system comprises: using Norvigilance Co LtdIII 1st Strand cDNA Synthesis Kit,R312, the reaction system is shown in Table 6 below.
Table 6.
The reaction procedure: 1: cycling for 1 time at 25 ℃ for 5 min; program 2: cycling for 1 time at 55 ℃ for 10 min; program 3: and (3) cycling for 1 time at 95 ℃ for 1 min.
Multiplex amplification reaction:
reagent: NEB company Q5 2X Master mix (M0494L), the two primer sets were amplified separately and the reaction system was as shown in Table 7 below.
Table 7.
| Reagent name | Addition amount of |
| Q5 2x MasterMix | 12.5μL |
| Primer set 3/4 (10 uM) | 4μL |
| Nuclease-free water | 6μL |
| Reverse transcription product | 2.5μL |
| Totalizing | 25μL |
The reaction procedure: program 1: cycling for 1 time at 95 ℃ for 30 sec; program 2: cycling for 30 times at 95 ℃,15sec,60 ℃ for 3 min; maintained at 4 ℃.
The library construction was carried out using the method of Nextera XT Library Preparation Kit (product number: FC-131-1024) from Illumina, inc. and the specific procedure is as follows:
(1) Combining and supplementing the amplified products of the two primer groups 3 and 4 to a total volume of 100 mu L, and purifying with 180 mu L of purification magnetic beads;
(2) Adding the purified product of the previous round into ATM and TD components respectively for fragmentation;
(3) The fragmented sequences are amplified by Index to complete library construction;
(4) Library quality inspection is performed: by usingThe dsDNA HS Assay Kit double-chain fluorescence quantitative kit is used for library quantification, and the yield of qualified libraries is more than 50 ng.
The library was sequenced after construction and the on-press data were assembled using commercial bioinformatics assembly software (e.g., qiagen. Clc Genomics Workbench) to analyze whole genome coverage and sequence quality.
Experimental results of coverage:
table 8.
The results illustrate: samples S1, S2 were pooled and sequenced for whole genome capture using the comparative example protocol, which did not allow good coverage for whole genome capture without the optimized protocol. The genome coverage of the whole genome reaching 20% of the average depth was only 75% or less, and the sample S1 was only 53.3%. Compared with the primer group of the comparative example, the final scheme of screening and optimizing in the invention has strong whole-gene sequence coverage uniformity, and the whole genome coverage maps of the human metapneumovirus samples S1-S2 are respectively shown in figures 5-6.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The primer combination for sequencing analysis of human metapneumovirus whole genome enrichment is characterized by comprising 29 primer sequences, wherein the 29 primer sequences are shown as SEQ ID NO. 1-SEQ ID NO. 29.
2. Use of the primer combination of claim 1 for detecting human metapneumovirus.
3. A test kit comprising the primer combination of claim 1.
4. The kit according to claim 3, wherein the reaction system of the kit comprises a reverse transcription system and an amplification system.
5. The test kit of claim 4, wherein the amplification system comprises: reverse transcriptase, high fidelity enzyme, taq enzyme, amplification reaction solution and dNTPs.
6. The test kit of claim 3, wherein the primer combination has a final concentration of 300nM in the amplification system.
7. The test kit of claim 3, wherein the reverse transcription procedure of the test kit is: program 1: cycling for 1 time at 25 ℃ for 5 min; program 2: cycling for 1 time at 55 ℃ for 10 min; program 3: and (3) cycling for 1 time at 95 ℃ for 1 min.
8. The kit of claim 3, wherein the amplification procedure of the kit is as follows: program 1: cycling for 1 time at 95 ℃ for 30 sec; program 2: cycling for 30 times at 95 ℃,15sec,60 ℃ for 3 min; maintained at 4 ℃.
9. The test kit of any one of claims 3-8, wherein the test sample of the test kit is one or more of a nasopharyngeal swab, a pharyngeal swab, saliva, sputum, and alveolar lavage.
10. Use of the detection kit of any one of claims 3-9 for detecting human metapneumovirus.
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|---|---|---|---|---|
| CA2411264A1 (en) * | 2002-12-19 | 2004-06-19 | Guy Boivin | Sequences for detection and identification of the human metapneumovirus |
| US20080213749A1 (en) * | 2007-03-02 | 2008-09-04 | Melanie Feola | Compositions and methods for detecting human metapneumovirus |
| CN112575124A (en) * | 2021-01-19 | 2021-03-30 | 季华实验室 | Multiple primer group for respiratory tract infection virus detection and construction method thereof |
| CN116790815A (en) * | 2023-06-26 | 2023-09-22 | 郑州安图生物工程股份有限公司 | Kit for detecting metapneumovirus |
-
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- 2024-01-18 CN CN202410074438.8A patent/CN117646088A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2411264A1 (en) * | 2002-12-19 | 2004-06-19 | Guy Boivin | Sequences for detection and identification of the human metapneumovirus |
| US20080213749A1 (en) * | 2007-03-02 | 2008-09-04 | Melanie Feola | Compositions and methods for detecting human metapneumovirus |
| CN112575124A (en) * | 2021-01-19 | 2021-03-30 | 季华实验室 | Multiple primer group for respiratory tract infection virus detection and construction method thereof |
| CN116790815A (en) * | 2023-06-26 | 2023-09-22 | 郑州安图生物工程股份有限公司 | Kit for detecting metapneumovirus |
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| Title |
|---|
| HOOGEN BERNADETTE G. VAN DEN 等: "Analysis of the Genomic Sequence of a Human Metapneumovirus", VIROLOGY, vol. 295, 31 December 2002 (2002-12-31) * |
| TULLOCH， R.L.等: "An Amplicon-Based Approach for the Whole-Genome Sequencing of Human Metapneumovirus", VIRUSES, vol. 13, 18 March 2021 (2021-03-18), pages 1 * |
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| 陆柔剑 等: "人偏肺病毒TaqMan-MGB探针实时定量RT-PCR检测方法的建立及初步应用", 生物技术通讯, vol. 19, no. 2, 31 March 2008 (2008-03-31), pages 1 * |
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