Primer set and detection method for detecting different types of human rhinoviruses

Abstract

The invention discloses a primer set and a detection method for detecting different types of a human rhinovirus, and relates to the technical field of microbial gene detection, the rhinovirus is a single-chain RNA virus, and a 0bp-650bp non-coding region needs to be selected in 7000bp. The primer set and the detection method have the advantages that the primer set is designed for the human rhinovirus, various types of the human rhinovirus can be effectively detected, and the detection accuracy is high. According to the present invention, the pathogenic microorganisms are detected by using the ultra-high heavy PCR technology and the second-generation high-throughput sequencing technology, the library can be established by adding 10 ng of the sample, all the primers can be directly mixed for use, the concentration of the single primer does not need to be adjusted, the experiment operation is simple and convenient, the whole process operation can be completed within 5 h, the orthogonal combination between the Index can be performed by using the double-end Index, the Index diversity is enriched, and the detection efficiency is high; according to the method, the final library mixing quantity of each lane is increased, the Index hopping effect is effectively reduced, the data is accurately split, and the sequencing accuracy is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial gene detection, and in particular to a primer set and a detection method for detecting different types of human rhinoviruses. Background Art

[0002] Human rhinovirus (HRV) is one of the common pathogens that cause upper respiratory tract infections in humans, and can cause diseases such as the common cold, acute sinusitis, and acute otitis media. Since there are multiple types of human rhinovirus, and the pathogenicity and epidemiological characteristics of different types may vary, accurate detection of human rhinovirus typing is of great significance for the diagnosis, treatment, and prevention of diseases.

[0003] Although HRV infection is usually self-limited, meaning it heals on its own without special treatment, it can sometimes lead to serious complications, such as asthma, congestive heart failure, bronchiectasis, and cystic fibrosis. In addition, HRV infection is often co-infected with other respiratory viruses, which can aggravate symptoms, prolong the course of the disease, and increase the risk of complications.

[0004] Human rhinovirus can be separated into more than 100 serotypes according to the difference of viral surface proteins, which are mainly divided into three categories: Human rhinovirus A (HRV-A), Human rhinovirus B (HRV-B) and Human rhinovirus C (HRV-C). Typing and detection of HRV is of great significance for clinicians to guide medication and pathological research of rhinovirus infection.

[0005] The detection technologies for human rhinovirus are mainly divided into traditional culture methods, serum antibody detection methods and molecular biological methods. Traditional detection methods mainly focus on tissue culture and virus isolation, serological detection methods include immunofluorescence and enzyme-linked immunosorbent assay (ELISA), etc. Gene detection methods mainly use nucleic acid molecular hybridization technology, RT-PCR technology and gene sequencing technology;

[0006] Existing methods for detecting human rhinovirus have many shortcomings, such as low detection sensitivity, poor specificity, and inability to detect multiple types simultaneously. Therefore, it is urgent to develop an efficient and accurate detection method that can simultaneously detect multiple types of human rhinovirus. To this end, we propose a primer set and detection method for detecting different types of human rhinovirus. Summary of the invention

[0007] The purpose of the present invention is to provide a primer set and a detection method for detecting different types of human rhinovirus.

[0008] In order to solve the problems raised in the above background technology, the present invention provides the following technical solutions: a primer set and a detection method for detecting different types of human rhinovirus, wherein the rhinovirus is a single-stranded RNA virus, and a non-coding region of 0 bp-650 bp needs to be selected from 7000 bp. The specific operation steps of the primer set detection method are as follows:

[0009] Step 1. Rhinovirus is a single-stranded RNA virus, and most types have a total length of more than 7000bp. The genome is composed of a 5' non-coding region (5'-untranslated region, UTR) from 0-650bp, and the subsequent 650bp to the tail is the coding region, which mainly encodes the four structural proteins VP1, VP2, VP3, and VP4. The 5' non-coding region plays an important role in the replication and translation of the virus, and studies have shown that the virulence of rhinovirus is related to the 5' non-coding region. This region is relatively conserved among different types of rhinovirus, so the primers are designed in this region;

[0010] Step 2: Download the three common rhinovirus reference genomes HRV-A, HRV-B, and HRV-C from the NCBI website (https: / / www.ncbi.nlm.nih.gov / datasets / genome / ), and screen the genomes for completeness, obtaining 101 HRV-A genomes, 37 HRV-B genomes, and 51 HRV-C genomes;

[0011] Step 3: Slide the first 650 bp of 189 rhinovirus genomes to generate rhinovirus subsequences of different lengths (sequence length greater than 80 bp and less than 500 bp), count the number of occurrences of different subsequences in all rhinovirus genomes, and obtain the top 50 sequences with the highest occurrence frequency;

[0012] Step 4: Use blast (version 2.14.0) to align all kmer sequences to the nt library and remove kmers other than the rhinovirus gene sequence;

[0013] Step 5. Use the online version of MFEprimer (https: / / mfeprimer3.igenetech.com / spec) to evaluate the remaining kmer sequences and select suitable primer sequences based on the GC content, hairpin structure prediction, and dimer structure prediction calculated by the software. If there is no suitable sequence, the sequence of this step is appropriately trimmed.

[0014] As a further solution of the present invention: in step 2, the positive sample needs to contain 5 different types of rhinovirus samples, and the negative sample needs to contain 5 samples that do not contain rhinovirus.

[0015] As a further solution of the present invention: in the step three, the first 25 bp and the last 25 bp sequences (kmer sequences) of the leading subsequence are respectively targeted and the kmer sequences are marked as candidate primer sequences for rhinovirus.

[0016] As a further solution of the present invention: the specific steps of the detection method are as follows:

[0017] Step 1: Take 32.5 μL of magnetic beads in a centrifuge tube, add all the 2nd PCR amplification products, pipette or vortex to mix, and let stand at room temperature for 5 minutes;

[0018] Step 2: After a slight instant centrifugation, place the centrifuge tube in a magnetic rack and let it stand for 3 minutes. After the liquid is clear, carefully discard the supernatant to avoid absorbing the magnetic beads.

[0019] Step 3: Add 200 μL of freshly prepared 80% ethanol, let stand for 30 seconds, and discard the supernatant after the solution becomes clear;

[0020] Step 4: Repeat step c and wash twice with alcohol;

[0021] Step 5: Use a 10μL pipette to remove the residual ethanol, open the lid to dry, and let it stand until there is no obvious gloss on the surface of the magnetic beads;

[0022] Step 6. Add 22 μL Nuclease-Free Water, pipette or vortex to mix, and let stand at room temperature for 5 minutes.

[0023] Step 7. Place the centrifuge tube in a magnetic rack, wait for the solution to clarify, aspirate 20 μL of supernatant, transfer it to the prepared 1.5 mL tube, mark the sample, and the final library is obtained.

[0024] Step 8. Measure and record library concentration and fragment size*.

[0025] By adopting the above technical solution, compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. The present invention designs a primer set for human rhinovirus, which can effectively detect multiple types of human rhinovirus, uses ultra-high-weight PCR technology and second-generation high-throughput sequencing technology to detect pathogenic microorganisms, and can build a library by investing 10ng of sample. All primers can be directly mixed and used without adjusting the concentration of a single primer. The experimental operation is simple and the whole process can be completed within 5 hours. The double-ended index can perform orthogonal combination between indexes, enrich the index diversity, increase the final mixed library quantity of each lane, effectively reduce the index hopping effect, accurately split the data, and improve the sequencing accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Schematic diagram of the primer sequence before and after in the embodiment of the present invention;

[0028] Figure 2 Schematic diagram of the experimental steps in the embodiment of the present invention.

[0029] Figure 3 Schematic diagram of primer sequences in the embodiments of the present invention.

[0030] Figure 4 This is a schematic diagram of rhinovirus detection results in an embodiment of the present invention.

[0031] Figure 5 The figure is a schematic diagram of the process of screening candidate primer sequences for rhinovirus in an embodiment of the present invention. DETAILED DESCRIPTION

[0032] The specific embodiments of the present invention are further described below in conjunction with the accompanying drawings. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] As attached Figure 1 -Attached Figure 4 As shown, the present invention is a primer set and detection method for detecting different types of human rhinovirus. The rhinovirus is a single-stranded RNA virus, and it is necessary to select a non-coding region of 0bp-650bp in 7000bp. The specific operation steps of the primer set detection method are as follows:

[0034] Step 1. Rhinovirus is a single-stranded RNA virus, and most types have a total length of more than 7000bp. The genome is composed of a 5' non-coding region (5'-untranslated region, UTR) from 0-650bp, and the subsequent 650bp to the tail is the coding region, which mainly encodes the four structural proteins VP1, VP2, VP3, and VP4. The 5' non-coding region plays an important role in the replication and translation of the virus, and studies have shown that the virulence of rhinovirus is related to the 5' non-coding region. This region is relatively conserved among different types of rhinovirus, so the primers are designed in this region;

[0035] Step 2: Download the three common rhinovirus reference genomes HRV-A, HRV-B, and HRV-C from the NCBI website (https: / / www.ncbi.nlm.nih.gov / datasets / genome / ), and screen the genomes for completeness, obtaining 101 HRV-A genomes, 37 HRV-B genomes, and 51 HRV-C genomes;

[0036] Step 3: Slide the first 650bp of 189 rhinovirus genomes to generate rhinovirus subsequences of different lengths (sequence length greater than 80bp and less than 500bp), count the number of occurrences of different subsequences in all rhinovirus genomes, obtain the top 50 sequences with the highest frequency of occurrence, and target the first 25bp and the last 25bp sequences (kmer sequences) of the top subsequences, and mark the kmer sequences as candidate primer sequences for rhinoviruses;

[0037] Step 4: Use blast (version 2.14.0) to align all kmer sequences to the nt library and remove kmers other than the rhinovirus gene sequence;

[0038] Step 5. Use the online version of MFEprimer (https: / / mfeprimer3.igenetech.com / spec) to evaluate the remaining kmer sequences and select suitable primer sequences based on the GC content, hairpin structure prediction, and dimer structure prediction calculated by the software. If there is no suitable sequence, the sequence of this step is appropriately trimmed.

[0039] In one embodiment of the present invention: Step 1, take 32.5 μL of magnetic beads in a centrifuge tube, add all 2ndPCR amplification products, pipette or vortex to mix, and let stand at room temperature for 5 minutes;

[0040] Step 2: After a slight instant centrifugation, place the centrifuge tube in a magnetic rack and let it stand for 3 minutes. After the liquid is clear, carefully discard the supernatant to avoid absorbing the magnetic beads.

[0041] Step 3: Add 200 μL of freshly prepared 80% ethanol, let stand for 30 seconds, and discard the supernatant after the solution becomes clear;

[0042] Step 4: Repeat step c and wash twice with alcohol;

[0043] Step 5: Use a 10μL pipette to remove the residual ethanol, open the lid to dry, and let it stand until there is no obvious gloss on the surface of the magnetic beads;

[0044] Step 6. Add 22 μL Nuclease-Free Water, pipette or vortex to mix, and let stand at room temperature for 5 minutes.

[0045] Step 7. Place the centrifuge tube in a magnetic rack, wait for the solution to clarify, aspirate 20 μL of supernatant, transfer it to the prepared 1.5 mL tube, mark the sample, and the final library is obtained.

[0046] Step 8. Measure and record library concentration and fragment size*.

[0047] Embodiment 1

[0048] Screening of candidate primer sequences

[0049] Rhinovirus is a single-stranded RNA virus, and most of the subtypes have a total length of more than 7000bp. The genome is composed of a 5'-untranslated region (UTR) from 0-650bp, and the subsequent 650bp to the tail is the coding region, which mainly encodes four structural proteins: VP1, VP2, VP3, and VP4. The 5'-untranslated region plays an important role in the replication and translation of the virus, and studies have shown that the virulence of rhinovirus is related to the 5'-untranslated region. This region is relatively conservative among different subtypes of rhinovirus, so primers are designed in this region.

[0050] Three common rhinovirus reference genomes, HRV-A, HRV-B, and HRV-C, were downloaded from the NCBI website (https: / / www.ncbi.nlm.nih.gov / datasets / genome / ), and the genomes were screened as complete, resulting in 101 HRV-A genomes, 37 HRV-B genomes, and 51 HRV-C genomes.

[0051] The first 650bp of 189 rhinovirus genomes were slid to generate rhinovirus subsequences of different lengths (sequence length greater than 80bp and less than 500bp), and the number of occurrences of different subsequences in all rhinovirus genomes was counted to obtain the top 50 sequences with the highest frequency of occurrence. The first 25bp and the last 25bp sequence (kmer sequence) of the leading subsequence were respectively targeted and the kmer sequence was marked as a candidate primer sequence for rhinovirus. The screening process is shown in the attached figure. Figure 5 As shown:

[0052] All kmer sequences were aligned to the nt library using blast (version 2.14.0), and kmers other than those aligned to rhinovirus gene sequences were removed.

[0053] The remaining kmer sequences were evaluated using the online version of MFEprimer (https: / / mfeprimer3.igenetech.com / spec), and appropriate primer sequences were selected based on the GC content, hairpin structure prediction, and dimer structure prediction calculated by the software. If there was no suitable sequence, the sequence for this step was appropriately trimmed.

[0054] Primer sequences

[0055] The primers screened in the above steps were aligned to the rhinovirus genome using bwa software (https: / / github.com / lh3 / bwa, version 0.7.18), respectively, using IGV software (https: / / www.igv.org / ).

[0056] Embodiment 2

[0057] cDNA one-strand synthesis

[0058] Preparation of mixed solution:

[0059]

[0060] Mix by pipetting or vortexing and centrifuge briefly.

[0061] Set the PCR instrument parameters, place the reaction solution on the PCR instrument, the reaction system is 20μL, and run the program:

[0062]

[0063]

[0064] Note: * The reaction time of this step can be adjusted appropriately according to actual needs to help increase the yield. The cDNA product is recommended to be used immediately in subsequent experiments.

[0065] 1st PCR

[0066] Prepare the mixture according to the following table:

[0067]

[0068] Mix by pipetting or vortexing and centrifuge briefly.

[0069] Set the PCR instrument parameters, place the reaction solution on the PCR instrument, the reaction system is 25μL, and run the program:

[0070]

[0071]

[0072] Note: #*Based on actual needs, different sample input amounts, etc., the conditions of these two steps can be appropriately changed.

[0073] 1st post-PCR purification

[0074] Remove the reaction system from the PCR instrument, centrifuge briefly, and place it in a centrifuge tube plate rack.

[0075] Add 20 μL of magnetic beads to the centrifuge tube in step a, mix by pipetting or vortexing, and incubate at room temperature for 5 minutes.

[0076] Centrifuge the tube briefly and place it on a magnetic rack. When the solution is clear, carefully remove the supernatant.

[0077] Keep the centrifuge tube in the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0078] Repeat step d. After removing the supernatant, centrifuge the tube briefly and place it on a magnetic rack to adsorb the magnetic beads. After the solution is clarified, use a 10 μL pipette to further remove the residual ethanol.

[0079] Keep the centrifuge tube in the magnetic rack all the time. When the surface of the magnetic beads has no obvious gloss, add 22μL Nuclease-Free Water, pipette or vortex to mix, and let stand at room temperature for 5 minutes.

[0080] Place the centrifuge tube in a magnetic rack, wait for the solution to become clear, and then pipette 20 μL of the solution for the next step.

[0081] 2nd PCR amplification

[0082] Prepare the mixture according to the following table:

[0083]

[0084] Mix by pipetting or vortexing and centrifuge briefly.

[0085] Set the PCR instrument parameters as follows, place the reaction solution on the PCR instrument, the reaction system is 50μL, and run the program:

[0086]

[0087] *The number of cycles can be changed appropriately according to actual needs.

[0088] Purification after 2nd PCR

[0089] Take 32.5 μL of magnetic beads in a centrifuge tube, add all the 2nd PCR amplification products, pipette or vortex to mix, and let stand at room temperature for 5 minutes.

[0090] After a slight instant centrifugation, place the centrifuge tube in a magnetic rack and let it stand for 3 minutes. After the liquid becomes clear, carefully discard the supernatant to avoid attracting the magnetic beads.

[0091] Add 200 μL of freshly prepared 80% ethanol, let stand for 30 seconds, and discard the supernatant after the solution becomes clear.

[0092] Repeat step c, washing with alcohol a total of two times.

[0093] Use a 10 μL pipette to remove the residual ethanol, open the lid and dry, and let it stand until there is no obvious gloss on the surface of the magnetic beads.

[0094] Add 22 μL Nuclease-Free Water, pipette or vortex to mix, and let stand at room temperature for 5 min.

[0095] Place the centrifuge tube in a magnetic rack, wait for the solution to become clear, aspirate 20 μL of the supernatant, transfer it to the prepared 1.5 mL tube, mark the sample, and the final library is obtained.

[0096] Measure and record library concentration and fragment size*.

[0097] Note: *Library quality assessment should use bioanalytical equipment to detect the size and peak shape of the library fragments. The main peak of the library fragment is 280bp-350bp. The library should have an obvious main peak, which means that the library construction is successful and can be loaded onto the machine normally.

[0098] Sequencing

[0099] The sequencing was performed using the T7 sequencer from Shenzhen BGI Genomics Co., Ltd. Data analysis:

[0100] Basic quality control filtering to obtain high-quality sequences:

[0101] Filter the adapter sequences in the offline data;

[0102] Filter bases with sequencing quality values ​​less than 20;

[0103] Sequences with a length less than 50 bp were filtered out.

[0104] Remove human sources:

[0105] The above-retained reads were aligned to the human reference genome using bowtie2 software with default parameters, and the sequences that were not aligned to the human reference genome were retained. For the unaligned sequences, they were aligned to human mRNA and rRNA using hisat2 software with default parameters, and the unaligned sequences were retained again for subsequent microbial identification.

[0106] Microbial sequence alignment and identification:

[0107] a) Download high-quality reference genomes of each rhinovirus genotype from the NCBI website, and then use bowtie2-build to build an alignment index for the reference genome.

[0108] b) Use bowtie2 to align the unaligned sequences to the rhinovirus reference genome. Then use samtools to count the reads aligned to each rhinovirus typing and calculate the RPM of different rhinovirus typing, that is, the number of reads aligned to the typing per million reads;

[0109] For rhinovirus typing identification, when the number of RPMs on each rhinovirus typing comparison was <1000, rhinovirus was not detected;

[0110] When the RPM of each rhinovirus typing comparison is ≥ 1000, the rhinovirus typing with the largest number of RPMs in the comparison is recorded as the detected rhinovirus typing of the sample.

[0111] Test results:

[0112] In the actual detection of 5 real samples, after the data analysis step, the number of reads obtained is shown in the following table. According to the rhinovirus identification rules, the judgment result is completely consistent with the sample type.

[0113] The present invention designs primers for the 5'UTR region (conserved region) of multiple rhinovirus typing genome sequences, and simultaneously designs primers for the single identical region of multiple sequences in combination with the different rhinovirus typing genome sequences, and then forms multiple pairs of primers (primer sets) by moving the primers upstream and downstream. Human rhinovirus has large variability, and effective amplification can still be obtained when base mutations occur in different subtypes by means of primer sets. Finally, multiple typing of rhinovirus can be identified by sequence comparison, and multiple human rhinovirus typing can be effectively detected, and has good accuracy and specificity. The present invention has a wide range of application prospects, is applicable to hospitals and professional testing institutions, and can be well targeted at the prevention and control of human rhinovirus infection, reducing disease transmission and spread.

[0114] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0115] The above contents are merely examples and explanations of the present invention. Various modifications or additions to the specific embodiments described or replacements in similar ways by technicians in the technical field shall fall within the protection scope of the present invention as long as they do not deviate from the invention or exceed the scope defined by the claims.

[0116] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A primer set for detecting different types of human rhinovirus, characterized in that: The rhinovirus is a single-stranded RNA virus, and it is necessary to select a non-coding region of 0 bp-650 bp in 7000 bp. The specific operation steps of the primer set detection method are as follows: Step 1. Rhinovirus is a single-stranded RNA virus. Most types have a total length of more than 7000 bp. The genome is composed of a 5' non-coding region (UTR) from 0-650 bp, and the next 650 bp to the tail is the coding region, which mainly encodes four structural proteins: VP1, VP2, VP3, and VP4. The 5' non-coding region plays an important role in viral replication and translation, and studies have shown that the virulence of rhinovirus is related to the 5' non-coding region. This region is relatively conserved among different types of rhinovirus, so primers are designed in this region; Step 2: Download three common rhinovirus reference genomes, HRV-A, HRV-B, and HRV-C, from the NCBI website, and simultaneously screen the genomes for completeness, obtaining 101 HRV-A genomes, 37 HRV-B genomes, and 51 HRV-C genomes; Step 3: Slide the first 650 bp of 189 rhinovirus genomes to generate rhinovirus subsequences of different lengths (sequence length greater than 80 bp and less than 500 bp), count the number of occurrences of different subsequences in all rhinovirus genomes, and obtain the top 50 sequences with the highest occurrence frequency; Step 4: Use blast (version 2.14.0) to align all kmer sequences to the nt library and remove kmers other than the rhinovirus gene sequence; Step 5. Use the online version of MFEprimer to evaluate the remaining kmer sequences. Select appropriate primer sequences based on the GC content, hairpin structure prediction, and dimer structure prediction calculated by the software. If there is no suitable sequence, the sequence for this step is appropriately trimmed.

2. A primer set for detecting different types of human rhinovirus according to claim 1, characterized in that: In step 2, the positive samples need to contain 5 different types of rhinovirus samples, and the negative samples need to contain 5 samples that do not contain rhinovirus.

3. A primer set for detecting different types of human rhinovirus according to claim 1, characterized in that: In the step three, the first 25 bp sequence and the last 25 bp sequence (kmer sequence) of the leading subsequence are respectively targeted and the kmer sequence is marked as a candidate primer sequence for rhinovirus.

4. A detection method for a primer set for detecting different types of human rhinovirus according to any one of claims 1 to 3, characterized in that: The specific steps of the detection method are as follows: Step 1: Take 32.5 μL of magnetic beads in a centrifuge tube, add all the 2nd PCR amplification products, pipette or vortex to mix, and let stand at room temperature for 5 minutes; Step 2: After a slight instant centrifugation, place the centrifuge tube in a magnetic rack and let it stand for 3 minutes. After the liquid is clear, carefully discard the supernatant to avoid absorbing the magnetic beads. Step 3: Add 200 μL of freshly prepared 80% ethanol, let stand for 30 seconds, and discard the supernatant after the solution becomes clear; Step 4: Repeat step c and wash twice with alcohol; Step 5: Use a 10μL pipette to remove the residual ethanol, open the lid to dry, and let it stand until there is no obvious gloss on the surface of the magnetic beads; Step 6. Add 22 μL Nuclease-Free Water, pipette or vortex to mix, and let stand at room temperature for 5 minutes. Step 7. Place the centrifuge tube in a magnetic rack, wait for the solution to clarify, aspirate 20 μL of supernatant, transfer it to the prepared 1.5 mL tube, mark the sample, and the final library is obtained. Step 8. Measure and record library concentration and fragment size*.

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