A human metapneumovirus whole genome enrichment kit for sequencing analysis

Abstract

The present invention discloses a human metapneumovirus whole genome enrichment kit for sequencing analysis, and relates to the field of biotechnology. The present invention provides a primer combination for enrichment of the human metapneumovirus whole genome for sequencing analysis, the primer combination comprising 29 primer sequences, and the 29 primer sequences are shown in SEQ ID NO:1-SEQ ID NO:29. The primer combination of the present invention adopts a shingled arrangement in the sequence, which better covers the whole genome sequence of the metapneumovirus. The primer set provided by the present invention performs capture and library construction sequencing on different samples, and the sequencing depth ≥1000× or more can still achieve a whole genome coverage of more than 99%, with very high coverage uniformity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a human metapneumovirus whole genome enrichment kit for sequencing analysis. Background Art

[0002] Human metapneumovirus (HMPV) is a common human respiratory pathogen. Its particles are pleomorphic, spherical, and fibrous under an electron microscope. HMPV belongs to the Pneumoviridae family, the Metapneumovirus genus, and is an enveloped single-stranded negative-sense RNA virus. HMPV contains 8 genes and 9 open reading frames. The proteins encoded mainly include genes for 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), and polymerase (L) subunit. HMPV has two genotypes, A and B, and four genotypes, A1, A2, B1, and B2. The A2 subtype is further divided into A2a and A2b gene clusters.

[0003] In 2001, Dutch scholars first detected HMPV from nasopharyngeal aspirate samples of infected children. HMPV is mainly transmitted through the respiratory system, and coughing and sneezing, close personal contact and touching virus contaminants can cause transmission. Generally speaking, the incubation period after infection is about 3-5 days, the detection rate is highest in winter and spring, and repeated infections are common. HMPV infection can also cause outbreaks, especially in children, causing symptoms such as hypoxemia, fever, cough, and wheezing.

[0004] There are currently three commonly used methods for detecting HMPV, including: antigen-antibody detection, viral cell culture, and reverse transcription polymerase chain reaction (RT-PCR). Viral cell culture technology requires high standards and takes a long time to culture. The cytopathic effect is difficult to identify and has low sensitivity. Commonly used clinical methods for HMPV antigen detection include immunofluorescence, immunochromatography, and enzyme immunoassay. However, these detection technologies can only identify pathogens, and cannot analyze the entire genome sequence of pathogens, nor can they track and trace the epidemic mutations of the virus in more depth.

[0005] Chinese patent CN101446592A discloses an indirect immunofluorescence detection kit for human metapneumovirus, which includes reagent A: mouse anti-human metapneumovirus monoclonal antibody: 1 ml of a solution with a volume percentage of 1% BSA of 1:50-20000; reagent B: 100 ml of phosphate buffer containing Tween 20; reagent C: FITC-labeled goat anti-mouse IgG antibody: 1 ml of a solution with a volume percentage of 1:10-1000 of reagent B; reagent D: 1 ml of Evans blue dye solution with a concentration of 0.01%. However, the sensitivity and specificity of the kit provided by the invention are inferior to RT-PCR. RT-PCR technology is the most sensitive and effective method for detecting HMPV infection, with high sensitivity and short reaction time.

[0006] The present invention has developed a complete set of primers that can cover the entire genome sequence of human metapneumovirus for downstream library construction and sequencing analysis, which is helpful to better monitor the process trends and mutations of human metapneumovirus in China to prevent sudden outbreaks. Summary of the invention

[0007] The purpose of the present invention is to provide a human metapneumovirus whole genome enrichment kit for sequencing analysis. The primer combination of the present invention better covers the whole genome sequence of the metapneumovirus and has high coverage uniformity.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] On the one hand, the present invention provides a primer combination for enrichment of the whole genome of human metapneumovirus for sequencing analysis, wherein the primer combination comprises 29 primer sequences, and the 29 primer sequences are shown in SEQ ID NO:1-SEQ ID NO:29.

[0010] SEQ ID NO: 1 is: AAAATGTCTCTTCAAGGGATTCACC.

[0011] SEQ ID NO:2 is: TGACTTGTCCCASTTTTTTAATTACTC.

[0012] SEQ ID NO: 3 is: CACAGATGAAGAAAAAGARGCTGC.

[0013] SEQ ID NO: 4 is: CTTTTTCTACYAGATCAACTTGAACAGC.

[0014] SEQ ID NO:5 is: GGGACAAGTMAARATGGAGTCCTA.

[0015] SEQ ID NO:6 is: TCATGTAGYACTATAACTGARGGATA.

[0016] SEQ ID NO:7 is: GGGACAARTAAAATGTCTTGGAAAGT.

[0017] SEQ ID NO:8 is: CCATRCTGATAGGRTGYCTTCCTGT.

[0018] SEQ ID NO:9 is: TCAARRGARTGCAACATCAACATATC.

[0019] SEQ ID NO: 10 is: CAGGAGTTTTGCTCATCTCCAT.

[0020] SEQ ID NO:11 is: GCTGATGGYYTRTCAATAATATCAGG.

[0021] SEQ ID NO:12 is: TTCCATTCRATTGTGTATATACAATGGCA.

[0022] SEQ ID NO:13 is: GCTGGAAAATAAGCAGAAATCAATG.

[0023] SEQ ID NO:14 is: GGATCCATTGTTATTTRTCYCTT.

[0024] SEQ ID NO:15 is: ATGCARCATGAAATAATGAARAAT.

[0025] SEQ ID NO:16 is: MAAGRGAYAAATAACAATGGATCC.

[0026] SEQ ID NO: 17 is: TTCTGAATATATARTATAATTCTGCAGCA.

[0027] SEQ ID NO: 18 is: CAACTGTTAACATGGAAAGATGTGATG.

[0028] SEQ ID NO: 19 is: AGAAGAAAGTTCYGATTTGATTTCCAT.

[0029] SEQ ID NO:20 is: TGATYTAGATCTYCARAGAATWATGGAAAT.

[0030] SEQ ID NO:21 is: CTGCTGTTCTATTKATATCACTTGTTAC.

[0031] SEQ ID NO:22 is: ACAACACTAATGAGAGAYCCTCAGG.

[0032] SEQ ID NO: 23 is: CACTCATTATRCTAATTCCRCAGCT.

[0033] SEQ ID NO:24 is: CWGTTCCAGCTTATAGRACAACAAATTA.

[0034] SEQ ID NO: 25 is: CGTGTRAGAGCATGTGCCAT.

[0035] SEQ ID NO:26 is: AGAATGTTCAGCAARGTYATGTTTGA.

[0036] SEQ ID NO:27 is: CACTGCTATTYTCATYTTRGAATTTTGTAT.

[0037] SEQ ID NO: 28 is: GATGCTTTATTRATAACWTTRTGTGATGCAGA.

[0038] SEQ ID NO: 29 is: ATCTCTGCATTYCCYAGGTTATCTAT.

[0039] In yet another aspect, the present invention provides use of the above primer combination in detecting human metapneumovirus.

[0040] Specifically, the above application is used for the diagnosis, genotyping and sample tracing of human metapneumovirus.

[0041] In yet another aspect, the present invention provides a kit comprising the above-mentioned primer combination.

[0042] Preferably, the reaction system of the kit includes a reverse transcription reaction system and an amplification system.

[0043] More preferably, the reverse transcription reaction system comprises: reverse transcriptase (Enzyme Mix), random hexamers, and reverse transcription reaction solution (RT Mix).

[0044] Still more preferably, the reverse transcription reaction system comprises:

[0045] Components Addition amount (ul) 10X RT Mix 2 Enzyme Mix 2 Random hexamers 1 RNA 10 <![CDATA[H 2 The]]> 5

[0046] Further preferably, the amplification system comprises: a high-fidelity enzyme, a Taq enzyme, a reaction amplification solution, and dNTPs (all included in 2x MasterMix).

[0047] Still more preferably, the amplification system comprises:

[0048]

[0049] Furthermore, the final concentration of the primer combination in the amplification system is 300 nM.

[0050] Preferably, the reverse transcription program of the kit is: program 1: 25°C, 5 min, 1 cycle; program 2: 55°C, 10 min, 1 cycle; program 3: 95°C, 1 min, 1 cycle.

[0051] Preferably, the amplification program of the kit is: program 1: 95°C, 30 sec, 1 cycle; program 2: 95°C, 15 sec, 60°C, 3 min, 30 cycles; maintain at 4°C.

[0052] Preferably, the kit is used for sequencing analysis.

[0053] Preferably, the test sample of the kit is one or more of nasopharyngeal swab, throat swab, saliva, sputum, and alveolar lavage fluid.

[0054] Preferably, the detection method of the kit comprises the following steps:

[0055] (1) extracting RNA from the sample to be tested and reverse transcribing the RNA into cDNA;

[0056] (2) PCR amplification of the reverse transcription product cDNA;

[0057] (3) Construct the library and sequence the PCR amplification products.

[0058] Preferably, the sequences of the primers used in the PCR amplification in step (2) are shown in SEQ ID NOs: 1-29.

[0059] Preferably, the steps of library construction described in step (3) are as follows:

[0060] (1) The amplification products of primer set 1 and primer set 2 were combined to a total volume of 100 μL, and then purified using 180 μL of purification magnetic beads;

[0061] (2) adding the purified products from the previous round to ATM and TD components for fragmentation;

[0062] (3) The fragmented sequences are further amplified through the Index to complete library construction;

[0063] (4) Perform library quality control.

[0064] In yet another aspect, the present invention provides use of the above-mentioned kit in detecting human metapneumovirus.

[0065] The beneficial effects of the present invention are:

[0066] The present invention provides a primer combination and a kit for enriching the whole genome of human metapneumovirus for sequencing. The kit can enrich all subtypes, and then connect the library construction and sequencing process, and the whole genome coverage rate reaches more than 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the full genome coverage map of human metapneumovirus sample S1.

[0068] Figure 2 This is the full genome coverage map of human metapneumovirus sample S2.

[0069] Figure 3 This is the full genome coverage map of human metapneumovirus sample S3.

[0070] Figure 4 This is the full genome coverage map of human metapneumovirus sample S4.

[0071] Figure 5 This is the whole genome coverage map of the comparative human metapneumovirus sample S1.

[0072] Figure 6 This is the whole genome coverage map of the comparative human metapneumovirus sample S2. DETAILED DESCRIPTION

[0073] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0074] Example 1

[0075] The primers for detecting human metapneumovirus are shown in Table 1 below.

[0076] Table 1 Primer sequences

[0077]

[0078]

[0079] The nucleotides at the degenerate sites in the above table are represented as R=A / G, Y=C / T, M=A / C, K=G / T, S=C / G, and W=A / T.

[0080] The coverage detection includes the following steps:

[0081] Whole-genome capture process:

[0082] Reverse transcription reaction:

[0083] System: Adopting Novezan III 1st Strand cDNA Synthesis Kit, R312, the specific reaction system is shown in Table 2 below.

[0084] Table 2.

[0085]

[0086]

[0087] Reaction program: Program 1: 25°C, 5 min, 1 cycle; Program 2: 55°C, 10 min, 1 cycle; Program 3: 95°C, 1 min, 1 cycle.

[0088] Multiplex Amplification Reaction:

[0089] Reagents: NEB Q5 2x MasterMix (M0494L), two primer sets for separate amplification, the specific reaction system is shown in Table 3 below.

[0090] Table 3.

[0091] Reagent name Addition amount Q5 2x MasterMix 12.5μL Primer set 1 / 2 (10uM) 4μL Nuclease-free water 6μL Reverse transcription product 2.5μL total 25μL

[0092] Reaction program: Program 1: 95°C, 30 sec, 1 cycle; Program 2: 95°C, 15 sec, 60°C, 3 min, 30 cycles; maintain at 4°C.

[0093] The library was constructed using Illumina's Nextera XT Library Preparation Kit (Cat. No.: FC-131-1024). The specific process is as follows:

[0094] (1) The amplification products of primer set 1 and primer set 2 were combined to a total volume of 100 μL, and then purified using 180 μL of purification magnetic beads;

[0095] (2) adding the purified products from the previous round to ATM and TD components for fragmentation;

[0096] (3) The fragmented sequences are further amplified through the Index to complete library construction;

[0097] (4) Perform library quality inspection: The dsDNA quantification kit (purchased from ThermoFisher, catalog number Q32854) was used for library quantification, and the output of a qualified library should be above 50 ng.

[0098] After library construction, sequencing is performed, and the data is assembled using commercial bioinformatics assembly software (such as Qiagen.CLCGenomics Workbench) to analyze the whole genome coverage and sequence quality.

[0099] Source of S1-S4 samples: Throat swab samples from patients infected with human metapneumovirus. After extraction with a commercial nucleic acid kit, the human metapneumovirus nucleic acid real-time fluorescence PCR detection kit (A3881, Zhuocheng Huisheng) was used to determine the sample nucleic acid Ct.

[0100] Experimental results of coverage:

[0101] Table 4.

[0102]

[0103] Results show that the whole genome length of samples S1-4 is 13350bp, and the coverage rate of the whole genome is more than 99% at a depth of 1000X or more. The coverage rate of the whole genome with an average depth of 20% or more is more than 90%, indicating that the whole sequence capture uniformity of this technical method is good. The whole genome coverage maps of human metapneumovirus samples S1-S4 are as follows: Figure 1-4 shown.

[0104] Comparative Example 1

[0105] The primers for detecting human metapneumovirus are shown in Table 5 below.

[0106] Table 5.

[0107]

[0108]

[0109] The comparative primer combination includes two sets of primers, primer set 3 and primer set 4.

[0110] The coverage detection method is the same as in Example 1.

[0111] Whole genome capture process:

[0112] Reverse transcription reaction:

[0113] System: Adopting Novezan III 1st Strand cDNA Synthesis Kit, R312, the reaction system is shown in Table 6 below.

[0114] Table 6.

[0115]

[0116]

[0117] Reaction program: 1: 25°C, 5 min, 1 cycle; program 2: 55°C, 10 min, 1 cycle; program 3: 95°C, 1 min, 1 cycle.

[0118] Multiplex Amplification Reaction:

[0119] Reagents: NEB Q5 2x MasterMix (M0494L), two primer sets for separate amplification, the reaction system is shown in Table 7 below.

[0120] Table 7.

[0121] Reagent name Addition amount Q5 2x MasterMix 12.5μL Primer set 3 / 4 (10uM) 4μL Nuclease-free water 6μL Reverse transcription product 2.5μL total 25μL

[0122] Reaction program: Program 1: 95°C, 30 sec, 1 cycle; Program 2: 95°C, 15 sec, 60°C, 3 min, 30 cycles; maintain at 4°C.

[0123] The library was constructed using Illumina's Nextera XT Library Preparation Kit (Cat. No.: FC-131-1024). The specific process is as follows:

[0124] (1) The amplification products of primer set 3 and primer set 4 were combined to a total volume of 100 μL, and then purified using 180 μL of purification magnetic beads;

[0125] (2) adding the purified products from the previous round to ATM and TD components for fragmentation;

[0126] (3) The fragmented sequences are further amplified through the Index to complete library construction;

[0127] (4) Perform library quality inspection: The dsDNA HS Assay Kit double-stranded fluorescence quantification kit is used for library quantification, and the output of qualified libraries should be above 50ng.

[0128] After library construction, sequencing is performed, and the data is assembled using commercial bioinformatics assembly software (such as Qiagen.CLCGenomics Workbench) to analyze the whole genome coverage and sequence quality.

[0129] Experimental results of coverage:

[0130] Table 8.

[0131]

[0132] Results: Samples S1 and S2 were captured and sequenced using the comparative solution. This unoptimized comparative solution could not capture and cover the whole genome well. The whole genome coverage rate of 20% average depth was only below 75%, and sample S1 was only 53.3%. Compared with the comparative primer set, the final solution screened and optimized in the present invention has a strong uniformity of whole genome sequence coverage. The whole genome coverage maps of human metapneumovirus samples S1-S2 are as follows: Figure 5-6 shown.

[0133] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer combination for enrichment of the whole genome of human metapneumovirus for sequencing analysis, characterized in that: The primer combination consists of 29 primer sequences, and the 29 primer sequences are shown in SEQ ID NO: 1-SEQ ID NO:

29.

2. A detection kit, characterized in that: The detection kit comprises the primer combination described in claim 1.

3. The detection kit according to claim 2, characterized in that The reaction system of the detection kit includes a reverse transcription system and an amplification system.

4. The detection kit according to claim 3, characterized in that The amplification system comprises: reverse transcriptase, high-fidelity enzyme, Taq enzyme, amplification reaction solution and dNTPs.

5. The detection kit according to claim 2, characterized in that The final concentration of the primer combination in the amplification system is 300 nM.

6. The detection kit according to claim 2, characterized in that The reverse transcription program of the detection kit is: program 1: 25°C, 5min, 1 cycle; program 2: 55°C, 10min, 1 cycle; program 3: 95°C, 1min, 1 cycle.

7. The detection kit according to claim 2, characterized in that The amplification program of the detection kit is as follows: program 1: 95°C, 30 sec, 1 cycle; program 2: 95°C, 15 sec, 60°C, 3 min, 30 cycles; maintain at 4°C.

8. The detection kit according to any one of claims 2 to 7, characterized in that: The detection sample of the detection kit is one or more of nasopharyngeal swab, throat swab, saliva, sputum, and alveolar lavage fluid.

Images