Primer pool and kit for detecting metapneumovirus whole genome and application of primer pool and kit
By designing a primer pool covering the entire genome, the problem of limited coverage of human metapneumovirus sequencing in existing technologies was solved, and the whole genome amplification and subtype identification of metapneumovirus were achieved. The sequencing depth and coverage were high, and the whole genome of metapneumovirus could be accurately detected.
Patent Information
- Application Number
- CN202510950470.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-10
- Publication Date
- 2025-10-17
AI Technical Summary
The existing technology has limited coverage of human metapneumovirus sequencing and cannot accurately detect multiple subtypes, which affects the accuracy of detection.
A primer pool for detecting the whole genome of metapneumovirus was designed, including a first primer pool and a second primer pool, with 45 pairs of primers covering the whole genome. The primers were divided into two primer pools using the shingled principle for whole genome amplification, covering more than 99.65% of the complete sequence.
The whole genome amplification of the metapneumovirus was achieved, which can accurately identify the A or B subtype with uniform coverage and a sequencing depth of 39,000× or 31,000×, covering 99.5% or more than 100% of the genome.
Smart Images
Figure BDA0005492482320000031 
Figure BDA0005492482320000041 
Figure BDA0005492482320000051
Abstract
Description
TECHNICAL FIELD
[0001] The present disclosure relates to the field of biotechnology, in particular to a primer pool for detecting full genome of metapneumovirus, a kit and application thereof. BACKGROUND
[0002] Human metapneumovirus (hMPV) is a single-stranded negative-sense RNA virus with envelope, belonging to the family Paramyxoviridae and genus Metapneumovirus, and its genome length is about 13.3 kb. hMPV is mainly transmitted by droplets and close contact with virus-contaminated objects. hMPV can cause acute respiratory infections in people of all ages, and is usually a mild self-limiting disease, but it is significantly harmful to children, the elderly and immunocompromised individuals. It often causes respiratory symptoms such as fever, cough, wheezing, and runny nose in children, and can progress to bronchiolitis and pneumonia in severe cases. In elderly and immunocompromised individuals, the disease will be more severe, and even life-threatening conditions such as respiratory failure can occur. Therefore, the etiological diagnosis of hMPV is particularly important for the prevention and control of infectious diseases in China, and the development of a full-genome targeted amplification kit has very important public health and economic significance.
[0003] At present, the sequencing of human metapneumovirus mainly designs primers targeting F protein or G protein genes, and performs targeted amplification and sequencing, and then constructs an evolutionary tree and analyzes the sequence, which makes the current sequencing of human metapneumovirus have limited coverage, and cannot accurately detect multiple subtypes of human metapneumovirus, which seriously affects the accuracy of human metapneumovirus.
[0004] DISCLOSURE
[0005] In order to solve the problems of the prior art, the embodiments of the present disclosure provide a primer pool for detecting full genome of metapneumovirus, a kit and application thereof. The technical solution is as follows:
[0006] In one aspect, the present disclosure provides a primer pool for detecting full genome of metapneumovirus, the primer pool comprising a first primer pool and a second primer pool, the first primer pool comprising a first primer to a 58th primer, and the second primer pool comprising a 59th primer to a 112th primer, the nucleotide sequences of the first primer to the 112th primer being sequentially shown in SEQ ID NO: 1 to SEQ ID NO: 112 in the sequence listing.
[0007] In another aspect, the present disclosure provides a kit for detecting full genome of metapneumovirus, the kit comprising the primer pool according to claim 1.
[0008] In yet another aspect, the present disclosure provides an application of a primer pool for detecting a full genome of a metapneumovirus, the application comprising: using the primer pool of claim 1 for detecting the full genome of the metapneumovirus.
[0009] Specifically, the detection sample is a nasopharyngeal swab, deep cough sputum or alveolar lavage fluid.
[0010] The technical solution provided by the embodiments of the present disclosure has the beneficial effects that: the present disclosure provides a primer pool for detecting a full genome of a metapneumovirus, according to the principle of imbrication, 45 pairs of primers are designed for the full genome of the metapneumovirus, and the 45 pairs of primers are divided into two primer pools, so that the full genome amplification of the metapneumovirus typing can be realized, more than 99.65% of the complete sequence of the metapneumovirus is covered, and the coverage is uniform. The method provided by the present disclosure can accurately identify the A or B subtype of the metapneumovirus. BRIEF DESCRIPTION OF DRAWINGS
[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present disclosure, and other drawings can also be obtained by those skilled in the art without creative labor.
[0012] Figure 1 is a sequencing depth statistical chart provided by the third embodiment of the present disclosure;
[0013] Figure 2 is a sequencing depth statistical chart provided by the fourth embodiment of the present disclosure. DETAILED DESCRIPTION
[0014] In order to make the purpose, technical solutions and advantages of the present disclosure clearer, the embodiments of the present disclosure will be further described in detail below with reference to the drawings.
[0015] Embodiment one
[0016] The present disclosure provides a primer pool for detecting a full genome of a metapneumovirus, the primer pool comprising a first primer pool and a second primer pool, the first primer pool comprising a first primer to a 58th primer, and the second primer pool comprising a 59th primer to a 112th primer, the nucleotide sequences of the first primer to the 112th primer being shown in the sequence table as SEQ ID NO: 1 to SEQ ID NO: 112 in order, and referring to Table 1.
[0017] Table 1 primer sequences and corresponding primer pools
[0018]
[0019]
[0020]
[0021]
[0022] The embodiment is based on the principle of imbricate, 45 pairs of primers are designed for the whole genome of metapneumovirus, and the primers are divided into two primer pools, and the metapneumovirus is amplified.
[0023] Example Two
[0024] The present disclosure provides a kit for detecting the whole genome of metapneumovirus, which comprises the primer pool provided in Example One.
[0025] Example Three
[0026] The present disclosure provides an application of a primer pool for detecting the whole genome of metapneumovirus, which comprises: using the primer pool provided in Example One to detect the whole genome of metapneumovirus.
[0027] The present embodiment detects a metapneumovirus sample provided by Hubei Center for Disease Control and Prevention.
[0028] RNA reverse transcription
[0029] Specifically, the metapneumovirus sample is reverse transcribed to obtain cDNA, and the reverse transcription amplification system is shown in Table 2.
[0030] Table 2 is the reverse transcription amplification system
[0031] Reagent Volume RT Supermix 3 μL Template RNA 12 μL Total volume 15 μL
[0032] The reverse transcription amplification procedure is shown in Table 3.
[0033] Table 3 is the reverse transcription amplification procedure
[0034] Temperature Time Cycles 25℃ 10 min 1 50℃ 10 min 1 85℃ 5 min 1 4℃ Hold
[0035] The cDNA is purified to obtain purified cDNA.
[0036] The purified cDNA is divided into two parts, and one of the purified cDNA is used for the first PCR amplification using the first primer pool provided in Example One to obtain the first amplification product; the amplification system of the first PCR amplification with a total volume of 25 μL includes: 2.5 μL of purified cDNA, 4 μL of 10 μM first primer pool, 12.5 μL of Hotstart hifi Mix and 6 μL of Nuclease-free water. The amplification procedure of the first PCR amplification is shown in Table 4.
[0037] Table 4 is the amplification procedure of the first PCR amplification
[0038]
[0039] A second PCR amplification of another purified cDNA was performed using the second primer pool provided in Example 1 to obtain a second amplified product. The amplification system for the second PCR amplification, per a total volume of 25 μL, included: 2.5 μL of purified cDNA, 4 μL of the 10 μM second primer pool, 12.5 μL of Hotstart HiFi Mix, and 6 μL of Nuclease-free water. The amplification procedure for the second PCR amplification was the same as that for the first PCR amplification provided in Table 4.
[0040] The first amplification product and the second amplification product were combined into a 1.5 mL centrifuge tube to obtain the whole genome amplified nucleic acid of the metapneumovirus for library construction.
[0041] Next-generation sequencing
[0042] The amplified nucleic acid of the whole genome of the metapneumovirus was purified using 0.8 volumes of magnetic beads. After purification, samples with a qubit quantification above 10 ng / μL could be directly used for subsequent library construction experiments. In this example, the Nextera XTDNA Library Prep Kit and MiniSeq Rapid Reagent Kit were used for library construction and sequencing. The data was assembled and analyzed using assembly software and compared with the database to obtain typing information. The analysis results are shown in Table 5.
[0043] Table 5 shows the assembly analysis results.
[0044] Category Number (bp) Proportion Relative to reference strain < 10x 21 0.16％ Relative to reference strain > 10x 13.1K 99.84％ Relative to reference strain > 50x 13.1K 99.83％ Relative to reference strain > 100x 13.1K 99.83％ Relative to reference strain > 200x 13.1K 99.82％
[0045] As shown in Table 5, the sequencing data covered more than 99.5% of the genome.
[0046] The sequencing depth is calculated based on the sequencing results. The statistical results are as follows: Figure 1 As shown by Figure 1 It can be seen that the average sequencing depth can reach 39000×.
[0047] Example 4
[0048] The present disclosure provides an application of a primer pool for detecting the whole genome of metapneumovirus, which includes: using the primer pool provided in Example 1 to detect the whole genome of metapneumovirus. This embodiment differs from Example 3 in that the detection object is different.
[0049] Specifically, this example tested the inactivated MPV strain isolated and cultured by the Hubei Provincial Center for Disease Control and Prevention.
[0050] Nucleic acid extraction
[0051] RNA from the inactivated strain was extracted using the EZ1&2Virus Mini Kit v2.0 (Cat. No. 955134) for the EZ1 Advanced XL fully automated nucleic acid purification instrument to obtain inactivated RNA. The RNA was reverse transcribed to obtain cDNA, which was purified to obtain purified cDNA. The purified cDNA was subjected to a first PCR amplification using the first primer pool provided in Example 1 to obtain a first amplification product. The first amplification product was subjected to a second PCR amplification using the second primer pool provided in Example 1 to obtain a second amplification product. The first amplification product and the second amplification product were combined to obtain a full-genome amplified nucleic acid of the metapneumovirus, which was used to construct a library. Next-generation sequencing was then performed. The specific process steps and parameters were all referenced to Example 3. The analysis results are shown in Table 6.
[0052] Table 6 shows the assembly analysis results.
[0053] Category Number (bp) Proportion Relative to reference strain < 10x 0 0.00％ Relative to reference strain > 10x 13.2K 100％ Relative to reference strain > 50x 13.2K 100％ Relative to reference strain > 100x 13.2K 100％ Relative to reference strain > 200x 13.2K 100％
[0054] As shown in Table 6, the sequencing data covered more than 100% of the genome.
[0055] The sequencing depth is calculated based on the sequencing results. The statistical results are as follows: Figure 2 As shown by Figure 2 It can be seen that the sequencing depth can reach 31000×.
[0056] Example 5
[0057] The present disclosure provides an application of a primer pool for detecting the whole genome of metapneumovirus, which includes: using the primer pool provided in Example 1 to detect the whole genome of metapneumovirus. This embodiment differs from Example 3 in that the detection object is different.
[0058] Specifically, this example tested 20 inactivated MPV strains isolated and cultured by the Hubei Provincial Center for Disease Control and Prevention, which were isolated and cultured from nasopharyngeal swabs, deep cough sputum, and alveolar lavage fluid.
[0059] Nucleic acid extraction
[0060] The RNA of the inactivated strain was extracted by using the EZ1 & 2 Virus Mini Kit v2.0, item number (955134) of the EZ1 Advanced XL full-automatic nucleic acid purifier to obtain the RNA of the inactivated strain. The RNA was reversely transcribed to obtain cDNA, which was divided into two parts. One part of the cDNA was subjected to first PCR amplification by using the first primer pool provided in Example 1 to obtain a first amplification product, and the other part of the cDNA was subjected to second PCR amplification by using the second primer pool provided in Example 1 to obtain a second amplification product. The first amplification product and the second amplification product were combined to obtain the full-genome amplification nucleic acid of the metapneumovirus, which was used for constructing a library. Then, second-generation sequencing was performed to obtain sequencing data, and the specific process steps and parameters were referred to those of Example 3.
[0061] The sequencing data was analyzed by using alignment software to obtain typing information, and then the genome was assembled by using assembly software to obtain consistent sequences. The sequencing depth was calculated according to the assembly results, and the calculation results are shown in Table 7.
[0062] Table 7 is the sequencing coverage statistics
[0063]
[0064]
[0065] As shown in Table 7, the coverage is more than 99.65%, and the typing of the MPV can be distinguished according to the amplification results of the primer pool.
[0066] The above description is only optional embodiments of the present disclosure, and does not limit the present disclosure. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.
Claims
1. A primer pool for detecting the whole genome of metapneumovirus, characterized in that: The primer pool includes a first primer pool and a second primer pool, the first primer pool includes the 1st primer to the 58th primer, the second primer pool includes the 59th primer to the 112th primer, and the nucleotide series of the 1st primer to the 112th primer are shown in SEQ ID NO: 1 to SEQ ID NO: 112 in the sequence listing.
2. A kit for detecting the whole genome of metapneumovirus, characterized in that: The kit comprises the primer pool according to claim 1.
3. An application of a primer pool for detecting the whole genome of metapneumovirus, characterized in that: The application includes: using the primer pool as described in claim 1 to detect the whole genome of metapneumovirus.
4. The use according to claim 3, characterized in that The test samples are nasopharyngeal swabs, deep cough sputum or alveolar lavage fluid.