CN114921588B - Targeting segment and primer set for detecting rhinovirus in sensing sample suitable for ONT sequencing platform and application thereof - Google Patents

Abstract

The application relates to the technical field of bioinformatics, and particularly provides a targeting segment and a primer set for detecting rhinovirus in a sample and application thereof, which are suitable for an ONT sequencing platform based on a raw signal primer design. The primers of the application can rapidly, efficiently, specifically and sensitively identify and distinguish different rhinoviruses from patients suffering from respiratory tract infection signs and symptoms.

Description

Targeting segment and primer set for detecting rhinovirus in sensing sample suitable for ONT sequencing platform and application thereof

Technical Field

The invention belongs to the technical field of bioinformatics analysis, and particularly relates to a targeting section and a primer group for detecting rhinoviruses in a sensitive sample, which are suitable for an ONT sequencing platform, and application thereof.

Background

Human Rhinovirus (HRVs) is the most common cause of human infections worldwide. It is a member of the enterovirus genus of the picornaviridae family, belonging to the picoviridae (diameter 30 nanometers), and the single-stranded RNA genome contains 7000 to 7500 nucleotides, enclosed in an icosahedral capsid. HRVs currently there are "HRV up to 150 to 170 genotypes in human infection (fig. 1), of which approximately 83 HRV-Sup>A, 32 HRV-B and 55 HRV-C types are found.

Rhinoviruses are common causes of the common cold, but also frequently occur in exacerbations of otitis mediSup>A, sinusitis, bronchitis, pneumoniSup>A, and asthmSup>A, where rhinovirus type Sup>A (HRV-Sup>A) and rhinovirus type C (HRV-C) are often associated with the occurrence of recurrent wheezing, and HRV-Sup>A (54.7%) and HRV-C (39.9%) account for the majority of HRV infections.

Differential diagnosis of rhinovirus infection is epidemiologically important. Recurrence of RV infection is very typical each year due to its great genetic diversity (> 150 serotypes). Specific recognition of rhinoviruses has had an impact on supportive therapies in patients, which becomes even more important when specific antiviral drugs are available. Nucleic acid amplification technology has replaced isolation of viruses in cell culture and has become the method of choice for detection of picornaviruses, in part because of its outstanding sensitivity, specificity and rapidity. Rhinoviruses have a conserved 5' non-coding region and several nearly identical sequence motifs, so that universal primers can be designed for amplification of reverse transcription-quantitative polymerase chain reaction. However, the sequence diversity of HRV genotypes presents challenges to develop molecular methods for efficiently detecting all genotypes. Most HRV RT-PCR assays target a conserved 5' non-coding region (NCR) that exhibits the greatest sequence homology in HRV genotypes. However, even in the 5' NCR, primers and probes must be designed with degenerate and modified bases or multiple oligonucleotides to amplify all HRV genotypes. The coding region of the viral capsid protein exhibits a high degree of heterogeneity compared to the rest of the HRV genome, leading to a wide range of antigen diversity and therefore often acting as a targeting region for HRV genotyping. The viral capsid consists of four capsid proteins VP1, VP2, VP3 and VP 4. The first three are present on the cell surface, while VP4 is present below the capsid. Among them, the VP1 and VP4+VP2 (VP 0) capsid regions are the most conserved viral capsid structures.

Although qualitative detection of HRV by RT-PCR is currently sufficient to determine HRV infection, accurate quantification of HRV RNA in clinical samples is required to investigate the relationship between HRV viral load and viral transmission, and patient symptoms and outcomes. Real-time RT-PCR analysis can be used to quantify the number of viral copies in a sample when accompanied by amplification of a standard curve (RT-qPCR). However, RT-qPCR analysis using a single HRV primer and probe set for quantification may not give accurate results for all genotypes due to low amplification efficiency caused by base mismatches between the primer and probe sequences and the specific viral sequences.

In view of this, the present invention has been proposed.

Disclosure of Invention

In order to solve the technical problems, the invention designs Sup>A pair of species-specific PCR primers for simultaneously detecting HRVs' NCR and VP genes respectively aiming at main infection species HRV-A and HRV-C by considering the difference between HRVs different strains as an amplification region through the own primer design system of the applicant according to the sequence information of various human rhinoviruses registered in GenBank, and establishes an effective amplification target sequence section. The specificity and sensitivity of the primer are tested, and the primer can be used for rapidly, conveniently and efficiently detecting the human rhinovirus, and has good specificity and high sensitivity. After the amplified product is obtained, the amplified product is combined with Nanopore sequencing and then analyzed, so that the different species of the human rhinovirus can be accurately identified and quantified.

It is therefore a first object of the present invention to provide a targeting segment for rhinovirus detection in an infection sample suitable for an ONT sequencing platform;

A second object of the present invention is to provide a primer combination for rhinovirus detection in an infected sample suitable for an ONT sequencing platform and a kit thereof;

a third object of the invention is to provide a method for rhinovirus detection in an infected sample based on the ONT sequencing platform.

Specifically, the technical scheme of the application is as follows:

the application firstly provides an inter-species specific, intra-species compatible target sequence suitable for ONT sequencing platform and used for detecting rhinovirus in an infection sample, wherein the target sequence comprises 5' NCR and VP gene sequences of human rhinovirus, and the target sequence comprises sequences shown in SEQ ID NO. 15-16.

The application also provides a primer combination suitable for detecting rhinovirus in an infection sample of an ONT sequencing platform, wherein the primer is aimed at a 5' NCR and VP gene sequence of human rhinovirus, and the primer combination comprises the following primers:

(1) HRV-Sup>A primers:

forward primer: CGGTAMCYTTGTRCGCCWGTT (SEQ ID NO. 1);

reverse primer: ARDGCATCSGGBARYTTCCACC (SEQ ID NO. 2)

(2) HRV-C primer:

Forward primer GKWCAAGYACTTCTGTTTCCC (SEQ ID NO. 3);

GGTGWTCKTGGAATCCATGCT (SEQ ID NO. 4) as a reverse primer;

Wherein M, Y, R, W, S, B, D are degenerate bases, M (A/C), K (G/T), Y (C/T), R (A/G), W (A/T), S (G/C), B (G/T/C), D (G/A/T) are degenerate base codes;

furthermore, the genome sequence of the target sequence section of the human rhinovirus amplified by the primer is shown as SEQ ID NO. 15-16.

Further preferably, the (1) HRV-A and (2) HRV-C primer sequences are as follows:

HRV-Sup>A primers:

forward primer F.1: CGGTAACTTTGTACGCCAGTT (SEQ ID NO. 5);

Reverse primer R.1: AGGGCATCCGGCAATTTCCACC (SEQ ID NO. 6);

forward primer F.2: CGGTAACCTTGTACGCCAGTT (SEQ ID NO. 7);

Reverse primer R.2: AGGGCATCCGGTAATTTCCACC (SEQ ID NO. 8);

Forward primer F.3: CGGTAACTTTGTACGCCAGTT (SEQ ID NO. 9);

reverse primer R.3: AGTGCATCCGGCAATTTCCACC (SEQ ID NO. 10);

HRV-C primer:

forward primer F.4: GGACAAGCACTTCTGTTTCCC (SEQ ID NO. 11);

reverse primer R.4: GGTGTTCTTGGAATCCATGCT (SEQ ID NO. 12);

Forward primer F.5: GGTCAAGTACTTCTGTTTCCC (SEQ ID NO. 13);

Reverse primer R.5: GGTGTTCGTGGAATCCATGCT (SEQ ID NO. 14).

Furthermore, the primer can specifically amplify human rhinovirus genes, and simultaneously can compatibly amplify human rhinoviruses with different sequences in the species, thereby ensuring species specificity and intra-species compatibility.

The application also provides a detection kit suitable for ONT sequencing platform and used for detecting rhinovirus in an infection sample, which comprises any one of the primers or the target sequence.

The application also provides application of any primer combination or target sequence in preparation of a detection kit suitable for ONT sequencing platform and used for detecting rhinovirus in an infection sample.

The application also provides application of any primer combination or target sequence in rhinovirus detection of an infected sample.

The application also provides an infection sample rhinovirus detection method based on the ONT sequencing platform, which is characterized by comprising the step of amplifying the infection sample DNA by utilizing any one of the primer combination or the kit.

Further, the method also includes sequencing and belief analysis steps.

Compared with the prior art, the primer for detecting the human rhinovirus has the following positive effects that:

(1) The primer combination designed by the application simultaneously considers the molecular characteristics of the 5'NCR and VP genes of the human rhinovirus, ensures the detection sensitivity by targeting the most conserved region 5' NCR, avoids missing detection, and simultaneously ensures the accurate identification of the intra-seed variation sequence by targeting the VP genes and combining with subsequent sequencing analysis; the method has the advantages that the intraspecies compatibility of the primer amplification interval is evaluated based on NCBI-ePCR software, taking HRV-A as an example, and the result shows that the HRV-A primer amplification interval covers more than 80% of the genotype of the HRV-A, the species specificity and the genotype compatibility can avoid the false positive or false negative problem of detection, and the method has absolute advantages in the field of detection of infection samples;

(2) The result of the human rhinovirus sensibility test based on the clinical real sample shows that the primer is applied to PCR detection of human rhinovirus genes, and compared with a third-party qPCR verification method, the positive consistency rate is 100 percent (11/11), and the target rate of amplified products in 11 samples is more than 93 percent;

(3) The primer of the application is based on the characteristic of long ONT sequencing reads length, the primer product length is about 1000bp-3000bp, and on the premise of selecting a conserved nucleotide sequence, the primer meets the basic principle of primer experiment design, and combines the ONT sequencing characteristic, and the primer product length is extended as far as possible, so that the primer is more suitable for an ONT platform, and can simultaneously perform quantitative and qualitative analysis.

In summary, the primer set of the application is a primer system suitable for ONT sequencing platform, suitable for rhinovirus detection of infected samples.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a contiguity (NJ) phylogenetic tree of relationships between all known HRV serotypes created based on VP gene sequences;

FIG. 2 primer binding ability assessment;

FIG. 3 is Sup>A specific assay for HRV-A primer amplification products;

FIG. 4 is a specific assay for HRV-C primer amplification products;

FIG. 5 is a diagram of a particular embodiment of the present invention for primer intra-species compatibility assessment;

FIG. 6 is a flow chart of a method of one particular embodiment of the present invention;

FIG. 7 shows the target rate of a sequenced sequence for the specificity assessment of primer amplification products in accordance with a specific embodiment of the present invention;

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the examples are some examples of the present invention but not all examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Partial term definition

Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the invention are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used herein, the terms "comprising," "including," "having," "containing," or "involving," are inclusive (inclusive) or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

The terms "about" and "substantially" in this invention mean the range of accuracy that one skilled in the art can understand yet still guarantee the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.

The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The application designs a targeted enrichment section suitable for an ONT sequencing platform and used for detecting rhinoviruses in an infection sample based on an own primer letter design analysis flow, and a corresponding primer combination, wherein the section and the primer combination solve the problems of specificity, compatibility and sensitivity of human rhinoviruses in the detection process of the infection sample:

in some embodiments, the primer amplifies a target sequence segment genomic sequence of human rhinovirus as shown in SEQ ID NO.15-16, with sequences 14 and 15 being base-degenerate sequences in view of sequence compatibility issues.

In some embodiments, the primer sequences are as follows:

(1) HRV-Sup>A primers: forward primer: CGGTAMCYTTGTRCGCCWGTT; reverse primer: ARDGCATCSGGBARYTTCCACC; (2) HRV-C primer: forward primer GKWCAAGYACTTCTGTTTCCC; reverse primer GGTGWTCKTGGAATCCATGCT; wherein M, Y, R, W, S, B, D are degenerate bases, and follow the degenerate base code table M (A/C), K (G/T), Y (C/T), R (A/G), W (A/T), S (G/C), B (G/T/C), D (G/A/T).

In some specific embodiments, it is preferred that the (1) HRV-A and (2) HRV-C primer sequences are as follows:

HRV-Sup>A primers: forward primer F.1: CGGTAACTTTGTACGCCAGTT; reverse primer R.1: AGGGCATCCGGCAATTTCCACC; forward primer F.2: CGGTAACCTTGTACGCCAGTT; reverse primer R.2: AGGGCATCCGGTAATTTCCACC; forward primer F.3: CGGTAACTTTGTACGCCAGTT; reverse primer R.3: AGTGCATCCGGCAATTTCCACC;

HRV-C primer: forward primer F.4: GGACAAGCACTTCTGTTTCCC; reverse primer R.4: GGTGTTCTTGGAATCCATGCT; forward primer F.5: GGTCAAGTACTTCTGTTTCCC; reverse primer R.5: GGTGTTCGTGGAATCCATGCT;

In some embodiments, the primer can specifically amplify human rhinovirus genes, can compatibly amplify human rhinoviruses with different sequences in species, ensures species specificity and intra-species compatibility, and has higher targeting rate. The target loading rate (ratio_ ontarget) is the Ratio of the rhinovirus related sequences obtained by sequencing to the primer targeting region.

Specific examples are as follows.

Example 1 design and optimization of primer sequences and target amplified regions

The application is used for detecting the human rhinovirus of the clinical infection sample, and false positive and false negative in the detection result need to be avoided in practice. Specifically, detection needs to be specific only for specific species of human rhinoviruses, other taxonomic or sequence homologous other viruses such as rhinovirus type B or other species in enterovirus cannot be amplified in the process, otherwise false positives of detection results are caused, and the primer design is required to have good 'inter-species' specificity; considering that the virus contains more than 150 serotypes, the sequences of different serotypes are different, for example, primer design cannot cover all serotypes, thus easily causing missed detection and further obtaining false negative results. Therefore, it is not easy to practice primer design for an infected sample, and the conventional primer design software or design thought cannot meet the actual requirement at all.

This example is based on applicant's early specific primer design patents (CN 112634983B, pathogen species specific PCR primer optimization design method) and facultative primer design patent (CN 111681711B, a facultative primer design screening method) for infection samples: the genomes of HRV-A and HRV-C are selected from NCBI RefSeq/GeneBank library, secondary structure and non-specific sequence intervals are eliminated through sequence comparison analysis, conserved nucleotide sequences are selected as amplification regions, and meanwhile, sup>A certain variable region exists in the middle, so that genotype differentiation exists in the targeted amplification regions. Meanwhile, whether the primer accords with the basic design principle of experiments or not is considered, for example, the primer cannot generate more than 4 continuous repeated bases, continuous complementation of more than 5 bases cannot generate among the primers, the 3' -end 5nt of the primer cannot generate more than 2 or 3 G+C, and the like, and the primer is designed.

The binding capacity of the designed primer to the template is further evaluated, first, the calcEndStability function in primer3 is used to calculate the free energy delta G value of the primer F/R and the target sequence of the target species and other species, if the delta G value of the other species is smaller than the delta G value of the target species, the primer is not evaluated by template binding, the primer is filtered out, and the evaluation result of the binding capacity of the primer to the template is shown in figure 2.

In addition, the amplification before sequencing detection is considered to be a multiplex amplification system, and the primer pressure is as low as possible on the basis of ensuring the detection accuracy, so that the problem of sequence compatibility of primer amplification is also ensured as much as possible in design, namely, all rhinoviruses are amplified through the minimum primers.

Finally, since the sequencing technology adopted by the application is the nanopore sequencing technology of ONT, which belongs to long fragment amplification, strict requirements are imposed on amplification primers and amplified fragments.

Through the above design and optimization, the application finally establishes that the primer amplifies the target region sequence as follows, the region comprises partial gene sequences of 5' NCR and VP of human rhinovirus (HRV-A: VP4/VP2; HRV-C: VP4-VP 1):

(1)HRV-A(SEQ ID NO.15)

CGGTRNNYTTRYRYRCCRGTTTKNNNNNYCNNNNNNNNNNNNNNNNNDYAAHNHTRNRARNNNNNNNNNHN

NNNNNNNNGDYCNRHAGNNNNNYRDYNNNNCCMNNNYRYNNNNGRHCRAGYACNYNYNGTNWYCCCNGGYN

NNNNNNNRATRNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNR

AARCCNNNNNNNHNGACADGRTGHGWNNAGYCNMBTGTGCTHRNHNTGHNTCCTCCGGCYCCYGAATGYGG

CTAAYCYNAANCCYRYWKCYRNNRYNYRHRWNCCADYRNRHNNNYRGTYGYAAYGVGHAABWRYNGGAYGG

RACCRACTACWTTGGRTGTCCGTNNNNNNGTTTCNYDNNNNNNWHNNNNNNNNNNNNNNNKYHYATGGTSA

CANNNNNNNNNNNNNNNNNNNTNNNNTGDBNYYATGGGYRCNCARGTNTCNMGRCARAAYGTWGGNACNCA

YWSHACNCARAAYDYNGTNDSNRRHGGNTCNARYYTNAAYTWYTTYAAYATHAAYTAYTTYAARGAYGCNG

CNTCNHVHGNNGCNTCHMRNYTDGANTTYWCNCADGAYCCNAGYAARTTYACNGAYCCNGTNAARGAYGTN

YTNDNNAARGRNRTNCCNACNYTNCARTCNCCNWSNGTNGARGCNTGYGGNTAYTCNGAYMGVHTNATNCA

RATHACNMGNGGNRAYWCNACNATNAYNTCNCARGANRYNGCHAAYGCNRTNRTNGSNTAYGSNVTNTGGC

CNNVNTAYYTNNVHNVNVVNSAYGCNDNNGCNATHGNYAARCCNWSNMRNCYNGAHRYNTCNDCNRAYMGV

TTYTAYACNYTDRVNAGYVDNNHNTGGNNNNNNNNNWSNHHDGGNTGGTGGTGGAARYTNCCNGAYGCNYT

(2)HRV-C(SEQ ID NO.16)

GRTCAAGCACTTCTGTTTCCCCGGRCYRAGTATCRAYAVRCYGNHYRYRYGGYTRARRGASAAARCGYYCG

TTAYCCGRCYARCTACTTCGRRAARYYYAGTAVCRCCRYBRAMRHBRCRGAGHGYTTCGYTCVRCACWNYC

CYMGTGTAGATCAGGYYGATGAGYCACYGYAWNCCYCAYGGGCGACCRTGGCRGTGRCTGCGYTGGCGGCC

TGCCYATGRNRHRMYYCRTRGGRCGCTYTRAWDYKGACATGGTGNGAARAGYCYAYTGAGCTAGYTRGYAR

TCCTCCGGCCCCTGAATGCGGCYAATCCYAACTGYGGAGCRCAYRCYYHCAANCCAGNGGGYRGTGYGTCG

TAAYGGGYAAYTCTGCRGCGRAACCGACTACTTTGGGTGYCCGTGTTTCHWBTYAYYYYYWYAYTGGCTGC

TWATGGTGACAATYRHDGARTTGTTACCATATAGCTATTGGATTGGCCATCCRGTKYNYAAYASAKCWRTH

RTHTAYYWRTTYRYTGGBTWYRTRCCWYTRDHHYYRARDTCYDYRDYYRCYCTHVRNYDYATHYTRAYYCT

YAAYAYRRYNRRRCATGGGYTCRCARGTDTCYRCNCARCGVTCYGGBTCNCAYGARAAYWYHAAYTCRGCY

ACNGARGGYTCYACYATWAAYTAYACYACYATYAAYTAYTAYAARSAYTCYTATGCHGCNACAGCRGGYAA

RCARAGYYTHARRCARGAYCCWGAYAARTTTRCNAAYCCYGTYAARGAYATYTTYACYGARATGGCDGCBC

CNYTRAARTCHCCRTCYGCYGARGCHTGTGGNTAYAGYGAYCGNGTRGCDCARYTHACYATYGGVAAYTCY

ACYATYACYACDCARGARGCRGCDAANATBATAGTYGGYTATGGHGARTGGCCHTCNTAYTGYTCNGAYDH

YGAYGCYACMGCRGTRGAYAARCCVACDCGYCCDGAYGTYTCNGTVAAYAGRTTYTAYACRYTRGAYACYA

AAYTRTGGGARAARTCRTCYAARGGRTGGTAYTGGAARTTYCCDGAYGTRYTRACHGARAYYGGRGTYTTY

GGBCARAAYGCNCARTTYCAYTAYYTMTAYMGRTNRGGRTTYTGYATYCAYGTRCARTGYAAYGCYAGYAA

RTTYCAYCAAGGRGCRNTVYTRGTYGCYRTHCTHCCRGARTAYGTYATWGGRACRGTRGCRGGYGGNACAG

GRRYNGARRAYWSYCAYCCYCMYTAYAWRCARACHCARCCHGGYGCYGAYGGYTTYGARYTRCARCAYCCR

TAYGTRCTYGAYGCHGGVATYCCHATATCRCARYTNACDRTRTGCCCHCAYCARTGGATYAAYYTGMGSAC

YAAYAAYTGYGCYACAATAATAGTRCCRTAYATNAAYRCAYTRCCYTTYGAYTCYGCYYTGAACCAYTGYA

AYTTTGGNYTRYTRGTNGTGCCYRTYAGYCCDYTRGAYTWYGAYCARGGDGCRACNCCRGTNATYCCYNTH

ACNATHACWYTRGCYCCRATGTGYTCYGARTTYGCWGGYCTYASGCARGCRGTYACBCARGGBTTYCCYAC

HGARYYRAAACCNGGBACVAAYCARTTYYTRACYACYGAYGAYGGHGTYTCRGCDCCYATYCTRCCDAAYT

TYCAYCCYACYCCVTGNATYCAYATACCYGGTGARGTBAGRAMYYTRCTHGARYTRTGYCARGTDGARACY

ATYYTNGARGTYAMYAAYGTRCCYACNAAYGYHACHAGYYTRATGGARAGRYTRCGNTTYCCRGTVTCHGC

NCARGCVGGRAARGGYGARYTRTGYGCNGTRTTYAGRGCYGAYCCHGGRMGDRAYGGHCCVTGGCARTCNA

CVHTRYTRGGYCARYTGTGYGGNTAYTAYACHCARTGGTCNGGDTCRYTRGARGTBACHTTYATGTTYACY

GGRTCYTTYATGGCYACNGGYAARATGCTYATAGCHTAYACACYNCCDGGHGGYCCYYTRCCYAARGAYMG

RGCNACMGCVATGYTRGGYACVCAYGTYATMTGGGAYTTYGGRNTRCARTCNTCYGTYACYCTTGTDATWC

CATGGATYAGYAAYRCHCAYTAYAGRGYVCAYGCYCGVGVYGGDKWVTTYGAYTAYTAYACYACDGGNHTV

RTNASYATNTGGTAYCARACNAAYTAYGTSGTNCCHATWGGDGCDCCYAMYACWGCHTAYATMRTNGCDYT

RGCRGCRGCYCARRADAAYTTYACNATGAARYTDTGYAARGAYRCBRRKSVYATHNWRCARACRGSYWMYA

THCARGGRGAYMSVRTDGCRGATRTRATYGAVMRHWCYRTWRRVRAYMRHGTRARYMRVKCHNTBACYSVV

KYNYWDSYRVYRCCNACMGSHSMVRAYACDSARGYRAGYARYCAYMGVYTRGRYACBGGYRWDGTHCCRGC

RCTBCARGCYGCBGARAYHGGRGCDTCRTCNAAYRCHAGYGAYRARARYMTSATTGARACDMGVTGYGTNY

TBAAYYMNCAYWSYACNSMDGARACNRCYHTNGRBARYTTYTWYAGYMGDGCDGGNYTDGTNRGVRWSATM

RMHMTBCCYHYHRMRGGYACHMMBAAYMCRRATGGBTATGYHAAYTGGRAHRTWGAYWYRRYNGGHTAYGC

DCARMTGCGBMGRAARKKVGARNTRTTYACHTAYATGCGYTTTGAYGCDGARTTYACHTTYGTHGYRKSYA

MNCCYAMHGGBSNRKTWGTYCCVCARYTRCTBCARTAYATGTWTGTNCCVCCWGGNGCYCCBAARCCDRMN

TCYAGRGADTCNYTYGCHTGGCARACWGCYACHAAYCCHTCDGTBTTYGTBAARHTRWCDGAYCCBCCRSC

DCARGTBTCNGTNCCVTTYATGTCRCCHGCDAGYGCHTAYCARTGGTTYTAYGAYGGDTAYCCCACVTTYG

GWGARCAYMWVCARGMRAAWGAYCTHGADTANGGVSMRTGYCCNAAYAAYATGATGGGYACNTTYWSVRTD

MGRRCHGTDGGRWSHKHVMARTCHMMRYAYYCHHTDRYNVTNAGRRTWTAYATGAGRATSAARCAYGTYAGRGCRTGGATMCCHMGMCCDMT。

Wherein M, K, Y, R, W, S, B, D, H, V, N are degenerate bases, and follow a degenerate base code table, M (A/C), K (G/T), Y (C/T), R (A/G), W (A/T), S (G/C), B (G/T/C), D (G/A/T), H (A/G/T), V (A/G/C), N (A/C/G/T).

The primer sequences for the final design and screening were as follows:

(1) HRV-Sup>A primers:

forward primer: CGGTAMCYTTGTRCGCCWGTT (SEQ ID NO. 1);

reverse primer: ARDGCATCSGGBARYTTCCACC (SEQ ID NO. 2);

(2) HRV-C primer:

Forward primer GKWCAAGYACTTCTGTTTCCC (SEQ ID NO. 3);

GGTGWTCKTGGAATCCATGCT (SEQ ID NO. 4) as a reverse primer;

wherein M, Y, R, W, S, B, D are degenerate bases, M (A/C), K (G/T), Y (C/T), R (A/G), W (A/T), S (G/C), B (G/T/C), D (G/A/T) are degenerate base codes;

preferably, the (1) HRV-A and (2) HRV-C optimal primer sequences are selected as follows:

HRV-Sup>A primers:

forward primer F.1: CGGTAACTTTGTACGCCAGTT (SEQ ID NO. 5);

Reverse primer R.1: AGGGCATCCGGCAATTTCCACC (SEQ ID NO. 6);

forward primer F.2: CGGTAACCTTGTACGCCAGTT (SEQ ID NO. 7);

Reverse primer R.2: AGGGCATCCGGTAATTTCCACC (SEQ ID NO. 8);

Forward primer F.3: CGGTAACTTTGTACGCCAGTT (SEQ ID NO. 9);

reverse primer R.3: AGTGCATCCGGCAATTTCCACC (SEQ ID NO. 10);

HRV-C primer:

forward primer F.4: GGACAAGCACTTCTGTTTCCC (SEQ ID NO. 11);

reverse primer R.4: GGTGTTCTTGGAATCCATGCT (SEQ ID NO. 12);

Forward primer F.5: GGTCAAGTACTTCTGTTTCCC (SEQ ID NO. 13);

Reverse primer R.5: GGTGTTCGTGGAATCCATGCT (SEQ ID NO. 14).

Example 2HRV primer specificity evaluation

The focus of this patent is that the proposed primers to detect HRV are able to specifically target this species, but not amplify other kinds of viral fragments. The specific description is as follows:

1. the NCBI functional network was logged in and the Primer-BLAST interface was opened.

2. The specific preferred primer sequence designed in example 1 was entered in the "PCR PARAMETERS" region and the target database of the verification primer was selected as nr in the "PRIMER PAIR SPECIFICITY CHECKING PARAMETERS" region.

3. The test results show that the input primers can specifically amplify HRV-A and HRV-C without crossing sequences of other virus species, and the existence of high species specificity is proved, and the specific results are shown in figures 3 and 4.

Example 3 compatibility assessment of different genotypes

The degenerate primers designed in the invention amplify different genotypes or serotypes of a particular species of rhinovirus, and the intraspecies compatibility of the primers is further evaluated by NCBI-ePCR, as follows:

1. Extracting a virus microorganism species sequence from the NT library according to the NCBI Taxonomy library, constructing a hash comparison library together with the human genome, further comparing F/R with the hash library respectively by using NCBI-ePCR software, obtaining a primer matching result, and extracting the sequence as a target product set;

2. And performing blast comparison on the target product set and the NT library, and filtering a comparison species result according to a bitscore threshold, wherein the bitscore threshold is max (bitscore) 0.95.

3. And (5) carrying out species and genotype statistics on the reserved comparison results.

Taking HRV-A as an example, the results show that the amplification region of the primers HRV-A F and HRV-A R covers 70 genotypes of HRV-A (FIG. 5), accounting for more than 80% of all genotypes of HRV-A (70/83).

In addition, in the sequencing analysis results of 11 clinical positive samples (see example 6), the rhinovirus detection result of each sample is of Sup>A specific species, and the simultaneous detection of HRV-A and HRV-C does not exist, which indicates that the primers respectively designed by the HRV-A and the HRV-C have species specificity, and the detection result contains different genotypes under the species, for example, the HRV-A can detect six genotypes of A16/A22/A34/A36/A38/A49 in 10 samples, and the above results indicate that the designed primers can be compatible with different genotypes in the species.

Example 4 human rhinovirus sequencing assay based on clinical samples

The focus of this patent lies in, and the primer that detects human rhinovirus that proposes can carry out specific amplification to the target interval to improve the sensitivity that rhinovirus detected. The overall application flow is shown in fig. 6, and is specifically described as follows:

1. Extracting RNA from 11 cases of human rhinovirus positive and 22 cases of human rhinovirus negative clinical samples respectively, wherein the sample type is alveolar lavage fluid (BALF) or sputum, and the judgment of positive/negative samples is based on the detection result of a third party detection method-rhinovirus nucleic acid detection kit (manufacturer: shanghai river);

2. adding HRV-A and HRV-C primers to construct an amplification reaction system, and then carrying out PCR amplification reaction;

3. after the amplification is completed, the amplified products are subjected to library construction, and further, the nanomore on-machine sequencing is carried out by referring to GridION using instructions;

4. firstly, quality control is carried out on the sequencing sequence, the long data of the nanopore is read, the sequence with low quality and too short length is filtered, and meanwhile, the joint sequence is removed;

5. After the quality control of the sequence, using blast and nt databases to carry out comparison so as to carry out species identification, and obtaining the comparison position of the sequence on a reference genome;

6. based on the result obtained in the last step, counting the sequence number (R) related to the human rhinovirus and the sequence number (Target-R) aligned to the Target area, and further obtaining the Target rate by calculating the ratio of the Target-R/R.

Example 5 evaluation of human rhinovirus sequencing assay results based on clinical negative samples

The invention further carries out the on-machine sequencing of the generated PCR reaction product by designing the PCR primer to amplify the conserved region of the human rhinovirus, counts the sequence number of the rhinovirus by analyzing the species attribute of the sequence, and counts the specificity of the primer based on the experimental result of 22 clinical negative samples. The specific embodiment is based on the procedure of example 4.

The results showed that the number of rhinovirus sequences obtained by Nanopore sequencing after PCR amplification using the designed primers was 0 for 22 negative samples, i.e., none of the 22 samples detected rhinovirus, with a specificity of 100% (22/22).

Example 6 evaluation of human rhinovirus sequencing assay results based on clinical Positive samples

The invention further establishes a library of PCR reaction products and performs on-machine sequencing on the Nanopore by designing a PCR primer to amplify a conserved region of human rhinovirus, and calculates the target rate of the amplified products and the sensitivity of the primer based on the experimental results of 11 clinical positive samples by analyzing species properties and alignment positions of the sequences. The specific embodiment is based on the procedure of example 4.

The statistics of rhinovirus sequences (Reads) and the target rate of 11 samples are shown in Table 1 and FIG. 7, and it can be seen that after PCR amplification using the designed primers, rhinoviruses can be detected in 11 samples at 1h of sequencing, and the positive coincidence rate is 100% (11/11). In addition, the coverage of rhinovirus related Reads in the targeted amplification area is counted, and the result shows that the target rate of all samples is above 93 percent and the median is 97.38 percent. Therefore, the primers designed by the invention can efficiently and specifically identify and distinguish rhinoviruses from respiratory tract infection patients.

TABLE 1.11 rhinovirus sequencing of HRV positive samples 1h detection results

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Sequence listing

<110> Jiangsu pioneer medical diagnosis Co., ltd

Nanjing pioneer medical inspection laboratory Co.Ltd

Beijing pioneer medical laboratory Co.Ltd

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Claims (3)

1. A primer combination suitable for rhinovirus detection in infection samples of ONT sequencing platform, characterized in that the primer combination is directed against the 5' ncr and VP gene sequences of human rhinovirus a-type and human rhinovirus C-type:

the gene sequences of the 5' NCR and VP of the A type of the human rhinovirus are shown in SEQ ID NO. 15; the gene sequences of the 5' NCR and VP of the C type of the human rhinovirus are shown in SEQ ID NO. 16;

The primer combination comprises an HRV-A primer and an HRV-C primer, and the sequences are respectively as follows:

HRV-Sup>A primers:

forward primer F.1: CGGTAACTTTGTACGCCAGTT (SEQ ID NO. 5);

Reverse primer R.1: AGGGCATCCGGCAATTTCCACC (SEQ ID NO. 6);

forward primer F.2: CGGTAACCTTGTACGCCAGTT (SEQ ID NO. 7);

Reverse primer R.2: AGGGCATCCGGTAATTTCCACC (SEQ ID NO. 8);

Forward primer F.3: CGGTAACTTTGTACGCCAGTT (SEQ ID NO. 9);

And a reverse primer R.3: AGTGCATCCGGCAATTTCCACC (SEQ ID NO. 10);

HRV-C primer:

forward primer F.4: GGACAAGCACTTCTGTTTCCC (SEQ ID NO. 11);

reverse primer R.4: GGTGTTCTTGGAATCCATGCT (SEQ ID NO. 12);

Forward primer F.5: GGTCAAGTACTTCTGTTTCCC (SEQ ID NO. 13);

And a reverse primer R.5: GGTGTTCGTGGAATCCATGCT (SEQ ID NO. 14).

2. A detection kit suitable for use in an ONT sequencing platform for the detection of rhinoviruses in an infected sample, comprising the primer combination of claim 1.

3. Use of a primer combination according to claim 1 for the preparation of a detection kit for use in an ONT sequencing platform and for the detection of rhinoviruses in an infected sample.