WO2023077491A1 - Combinaison de marqueurs mnp, combinaison de paire d'amorces et kit de virus respiratoire syncytial et son utilisation - Google Patents

Combinaison de marqueurs mnp, combinaison de paire d'amorces et kit de virus respiratoire syncytial et son utilisation Download PDF

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WO2023077491A1
WO2023077491A1 PCT/CN2021/129169 CN2021129169W WO2023077491A1 WO 2023077491 A1 WO2023077491 A1 WO 2023077491A1 CN 2021129169 W CN2021129169 W CN 2021129169W WO 2023077491 A1 WO2023077491 A1 WO 2023077491A1
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mnp
respiratory syncytial
syncytial virus
combination
marker
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PCT/CN2021/129169
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Chinese (zh)
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方治伟
高利芬
周俊飞
李论
李甜甜
彭海
陈利红
肖华峰
万人静
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江汉大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the embodiment of the present invention relates to the field of biotechnology, in particular to a respiratory syncytial virus MNP marker combination, primer pair combination, kit and application thereof.
  • Respiratory syncytial virus is an RNA virus that belongs to the genus Pneumovirus in the family Paramyxoviridae. There are two known subtypes, A and B. The virus is widespread worldwide, mainly through droplet transmission and close contact, and the susceptible populations are mainly children, the elderly and immunocompromised patients. After respiratory syncytial virus infection, infants and young children have severe symptoms, such as high fever, rhinitis, pharyngitis, and laryngitis, and then manifest as lower respiratory tract infections such as bronchiolitis and pneumonia.
  • Existing respiratory syncytial virus RSV detection technology is mainly through virus serotype identification, isolation and culture identification, indirect or direct immunofluorescence method and molecular detection technology for detecting genetic material on respiratory secretions, including PCR, nucleoside Acid hybridization and sequencing techniques. These technologies have their own advantages, but there are also one or more limitations in terms of time length, operational complexity, detection throughput, accuracy and sensitivity of detecting variants, and cost.
  • the isolation and identification of viruses is complicated and time-consuming; serotype identification, indirect or direct immunofluorescence methods are prone to cross-reactions, resulting in inaccurate typing, and cannot monitor mutations; one reaction of PCR detection technology is only for 1 to 2 markers of the virus The detection efficiency is low, and it is easy to fail the detection due to the mutation of the virus.
  • Metagenome sequencing technology is another technology for detecting respiratory syncytial virus, but it often includes a large amount of host sequencing data. When testing samples with low viral load, ultra-deep sequencing is especially required, resulting in high costs. Therefore, the development of a rapid, accurate, one-time high-throughput RSV detection and analysis method for the detection and typing of RSV is of great significance for the detection and prevention of RSV.
  • the invention develops a new type of molecular marker-MNP marker, and integrates super multiplex PCR amplification and high-throughput sequencing technology to analyze and detect MNP markers, and can type tens of thousands of MNP markers in hundreds of thousands of samples at one time, so as to realize the
  • the high-throughput, sensitive detection and accurate typing of the seven known respiratory syncytial viruses has the advantages of less sample requirements, accurate diagnostic results, saving data volume, and detecting mutations.
  • MNP markers refer to polymorphic markers caused by multiple nucleotides in an upper region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages: (1) rich allelic types, with 2 n allele types on a single MNP marker, higher than SSR and SNP markers; (2) strong species discrimination ability , only a small amount of MNP markers are needed to achieve species identification, reducing the detection error rate.
  • the MNP marker method based on ultra-multiplex PCR combined with next-generation high-throughput sequencing technology to detect MNP markers has the following advantages: (1) The output is base sequence, without parallel experiments, and a standardized database can be constructed for sharing; (2) High Efficiency, using sample DNA barcodes, breaking through the limitation of the number of sequencing samples, and can type tens of thousands of MNP markers in hundreds of samples at one time; (3) High sensitivity, using multiplex PCR to detect multiple targets at a time, avoiding single Target amplification failures lead to high false negatives and low sensitivity; (4) high accuracy, using a second-generation high-throughput sequencer to sequence the amplified product hundreds of times.
  • MNP marker and its detection technology can realize the classification and traceability of multi-allelic genotypes in populations, and has application potential in the identification of pathogenic microorganisms, fingerprint database construction, and genetic variation detection.
  • MNP marker method can realize the classification and traceability of multi-allelic genotypes in populations, and has application potential in the identification of pathogenic microorganisms, fingerprint database construction, and genetic variation detection.
  • MNP labeling there is no report on MNP labeling, and there is a lack of corresponding technology.
  • the purpose of the embodiment of the present invention is to provide a MNP marker combination of respiratory syncytial virus, a combination of primers, a kit and its application, which can identify and detect the variation of two subtypes of respiratory syncytial virus, and have the advantages of culture-free, multiple The effect of target, high throughput, high specificity, high sensitivity and fine typing.
  • the MNP marker refers to species-specific screening on the genome of respiratory syncytial virus, and has multiple nucleotides within the species The polymorphic genomic region, the MNP marker combination includes 15 markers from MNP-1 to MNP-15 on the respiratory syncytial virus reference sequence.
  • the specific nucleotide sequences of 15 markers are shown as SEQ ID NO.1-SEQ ID NO.15, wherein ID NO.16-SEQ ID NO.30 is the upper primer, ID NO.31-SEQ ID NO.45 is the lower primer.
  • Table 1 of the description further explains it.
  • the start and end positions of the MNP marker marked in Table 1 are determined based on the reference sequence corresponding to the same row of MNP in Table 1.
  • the multiple PCR primer pair combination includes 15 pairs of primers, and the specific primer sequences are as SEQ ID NO.16- Shown in SEQ ID NO.45, wherein ID NO.16-SEQ ID NO.30 is the upper primer ID NO.31-SEQ ID NO.45 is the lower primer.
  • each MNP-labeled primer includes an upper primer and a lower primer, as shown in Table 1 of the specification.
  • a detection kit for detecting the respiratory syncytial virus MNP marker combination includes the primer pair combination.
  • kit also includes a multiplex PCR master mix.
  • the MNP marker combination of the respiratory syncytial virus or the multiple PCR primer pair combination or the detection kit is provided in the identification of the respiratory syncytial virus and the construction of the MNP fingerprint database , Application in genetic variation detection.
  • the viral total RNA of the sample to be tested Utilize the commercial kit to carry out cDNA synthesis to the total RNA; Utilize the kit of the present invention to carry out the first round of multiplexing of the cDNA and the blank control PCR amplification, the number of cycles is not more than 25; after the amplification product is purified, add sample tags and next-generation sequencing adapters based on the second round of PCR amplification; quantify the second round of amplification products after purification; detection When there are multiple strains, high-throughput sequencing is performed by mixing the second-round amplification products in equal amounts; the sequencing results are compared to the reference sequence of RSV to obtain the number of detected sequences in the cDNA and genotype data.
  • the quality control scheme and the determination method use the RNA of RSV with known copy number as the detection sample, evaluate the sensitivity, accuracy and specificity of the kit for detecting RSV, and formulate the The quality control scheme and determination method for the detection of respiratory syncytial virus by the kit.
  • the detection of genetic variation of respiratory syncytial virus it includes the detection of genetic variation between strains and within strains.
  • the detection of genetic variation between strains includes using the above kit and method to obtain the genotype data of each of the 15 MNP markers of the strains to be compared. Through genotype comparison, analyze whether there are differences in the main genotypes of the strains to be compared on the 15 MNP markers. If there is a variation in at least one main genotype of the MNP marker in the strain to be compared, it is determined that there is a genetic variation between the two.
  • single-plex PCR can also be used to amplify the 15 markers of the strains to be compared, and then perform Sanger sequencing on the amplified products. After obtaining the sequence, the genotype of each MNP marker of the strain to be compared Compare.
  • MNP markers with inconsistent main genotypes there is variation among the strains to be compared.
  • a statistical model is used to determine whether a subgenotype other than the main genotype is detected in the MNP marker of the strain to be tested. If the strain to be tested has subgenotypes in at least one MNP marker, it is determined that there is genetic variation within the strain to be tested.
  • the genotype data of the MNP marker of the RSV identified from the sample is entered into the database file to form the MNP fingerprint database of the RSV; each identification
  • the MNP fingerprint database of the respiratory syncytial virus identify whether the respiratory syncytial virus in the sample and the strain in the database have a main genotype in the MNP marker (in a MNP marker with Genotypes supported by more than 50% of the sequenced fragments) differences, respiratory syncytial virus with major genotype differences in at least one MNP marker is a new variant type, and is included in the MNP fingerprint database.
  • the respiratory syncytial virus MNP fingerprint database constitutes the RSV reference sequence library; the genotype of the RSV in the sample to be tested is compared with the reference sequence library of the RSV, and the screen is genetically consistent or most For close strains, obtain the type of RSV in the sample to be tested. According to the comparison result with the reference sequence library, it is identified whether the RSV in the sample is an existing type or a new variant, so as to realize fine typing of the RSV.
  • MNP markers are mainly developed based on reference sequences, and large-scale identification of RSV from other species can be mined based on the resequencing data of the reported RSV representative races , MNP markers that are polymorphic and conserved on both sides of the respiratory syncytial virus species; MNP marker detection primers suitable for multiplex PCR amplification can be designed through the conserved sequences on both sides of the MNP marker; then according to the test results of the standard, A set of MNP markers with the largest polymorphism and high specificity, a primer combination with the best compatibility and a detection kit can be screened out.
  • the present invention has the following advantages:
  • the invention provides an MNP marker combination of respiratory syncytial virus, a primer pair combination, a kit and applications thereof.
  • the provided 15 MNP markers of respiratory syncytial virus and their primer combinations can be used for multiplex PCR amplification, and the next-generation sequencing platform can be used to sequence the amplified products, which meets the needs of high-throughput, high-efficiency, High-accuracy and high-sensitivity detection needs meet the requirements of RSV standard and sharable fingerprint data construction; accurate detection of genetic variation among RSV strains; identification of RSV homozygous and heterozygous To provide technical support for the scientific research, scientific monitoring and prevention of respiratory syncytial virus.
  • Figure 1 is a schematic diagram of MNP marker polymorphism
  • Fig. 2 is the screening and primer design flowchart of respiratory syncytial virus MNP marker
  • Fig. 3 is the detection flowchart of MNP mark
  • Example 1 The screening of respiratory syncytial virus MNP marker combination and the design of multiple PCR amplification primers
  • RSV-specific MNP markers were obtained through sequence alignment and comparison with the NCBI database.
  • the genome sequence information of representative races of the microbial species to be detected can also be obtained through high-throughput sequencing, where high-throughput sequencing can be whole genome or simplified genome sequencing.
  • high-throughput sequencing can be whole genome or simplified genome sequencing.
  • the genome sequences of at least 10 genetically representative isolates are generally used as references.
  • the step S1 specifically includes:
  • step lengths can also be used when screening on the reference genome with a window of 100-300 bp.
  • the step size is 1 bp, which is conducive to comprehensive screening.
  • the multiple PCR amplification primers labeled with MNP are designed by primer design software.
  • the primer design follows that the primers do not interfere with each other. All primers can be combined into a primer pool for multiple PCR amplification, that is, all designed primers can be used in one amplification reaction. normal expansion.
  • Respiratory syncytial virus RNA samples with known copy numbers provided by the Hubei Provincial Center for Disease Control and Prevention were reverse-transcribed into cDNA by a commercial reverse transcription kit, and then added to human genomic DNA to prepare 1000 copies/reaction
  • the template is detected by the detection method of multiplex PCR combined with next-generation sequencing using the designed primer combination.
  • Four repeated sequencing libraries were constructed, and according to the test results, 15 MNP markers and their detection primer pair combinations provided by the present invention were finally obtained, specifically as error! Reference source not found. shown.
  • the respiratory syncytial virus RNA sample with known copy number provided by the Hubei Provincial Center for Disease Control and Prevention was reverse-transcribed into cDNA by a commercial reverse transcription kit, and then added to human genomic DNA to prepare 1 copy/reaction, 10 copies/reaction, and 100 copies/reaction of RSV mock samples.
  • an equal volume of sterile water was set as a blank control.
  • the reproducibility and accuracy of the detection method were evaluated according to the number of sequenced fragments and markers of the MNP marker of RSV detected in the blank control and RSV nucleotide standards in 12 repeated experiments , Sensitivity, formulate thresholds and judgment standards for quality control system contamination and detection of target pathogens.
  • the sensitivity and stability evaluation of the MNP marker combination detection kit for detecting respiratory syncytial virus is shown in Table 2.
  • the kit can stably detect more than 5 MNP sites in a sample of 10 copies/reaction, while Detect up to 2 MNP sites in a small number of samples with 0 copies/reaction, the kit can clearly distinguish between 10 copies/reaction and 0 copies/reaction samples, with technical stability and detection as low as 10 copies/reaction sensitivity.
  • the reproducibility and accuracy of the MNP marker detection method for detecting respiratory syncytial virus were evaluated. Specifically, 12 sets of data of 100 copies of samples were compared in pairs. The results are shown in Table 3.
  • the kit can accurately detect RSV down to 10 copies/reaction.
  • the sequence aligned to RSV can be detected in 1 copy/reaction sample, covering at least 1 MNP marker.
  • sequence of respiratory syncytial virus was also detected in some blank controls. Due to the extreme sensitivity of the MNP marker detection method, data contamination during the detection process can easily lead to false positives. Therefore, the following quality control plan was formulated in this example.
  • the quality control plan is as follows:
  • the amount of sequencing data is greater than 4.5 million bases. The calculation is based on the fact that the number of MNP markers detected in each sample is 15, and the length of a sequencing fragment is 300 bases. Therefore, when the data volume is greater than 4.5 million bases, most samples can be guaranteed to cover each marker in one experiment. The number of sequencing fragments reaches 1000 times, ensuring accurate analysis of the base sequence of each MNP marker.
  • the noise index of the blank control P nc/Nc, wherein nc and Nc respectively represent the number of sequenced fragments and the total number of sequenced fragments of RSV in the blank control.
  • the signal index of the test sample S nt/Nt, wherein nt and Nt respectively represent the number of sequenced fragments and the total number of sequenced fragments of RSV in the test sample.
  • the average value of the signal-to-noise ratio of the sample of 1 copy of RSV and the blank control is 2.5, and the average value of the signal-to-noise ratio of the sample of 10 copies and the blank control is 31.1, therefore, this
  • the invention stipulates that when the signal-to-noise ratio is greater than 10 times, it can be judged that the contamination in the detection system is acceptable. In 12 sets of data with 10 copies/reaction, at least 5 MNP markers could be detected stably, accounting for 33.3% of the total markers.
  • the kit provided by the present invention can accurately and sensitively detect 10 copies/reaction of respiratory syncytial virus.
  • RNAs of influenza virus, measles virus, enterovirus EV71, coronavirus, influenza A virus, influenza B virus, influenza C virus, avian influenza virus and Zika virus were mixed together to prepare a mixed template, and a blank template was used as a control , using the kit provided by the present invention to detect the pathogen in the mixed template, and carry out 3 repeated experiments.
  • each strain can detect all 15 MNP markers (Table 5). Compare the fingerprints of the 6 strains in pairs, and the results are shown in Table 5 Wrong! Reference source not found. As shown, 1 (S-2) and 5 RSVs detected together with the same batch had some major genotype differences of some markers (Table 5), and there were variations among strains.
  • kits to identify the genetic variation between strains by detecting the MNP marker combination can be used to ensure the genetic consistency of the same named respiratory syncytial virus strains in different laboratories, thereby ensuring the comparability of research results, which is important for the respiratory tract.
  • the scientific research of syncytial virus is of great significance.
  • the diagnostic scheme can be considered according to whether differential markers affect drug resistance.
  • the authenticity evaluation of the secondary allelic type in this embodiment is carried out as follows: first, the allelic type with strand preference (the ratio of the number of sequencing sequences covered on the DNA double strand) is excluded according to the following rules: the strand preference is greater than 10 times, or more than 5 times different from the strand preference of the main allele type.
  • e max (n 1) and e max (n ⁇ 2) of 1.03% and 0.0994%, respectively, were obtained from the frequencies of all minor alleles detected at 930 homozygous MNP markers.
  • the nucleotides of the two strains with different genotypes shown in Table 5 are divided into the following eight ratios: 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/ 100, 5/100, and 7/100 were mixed to prepare artificial heterozygous samples, and each sample was tested 3 times to obtain a total of 24 sequencing data.
  • markers of heterozygous genotypes were detected in 24 artificial heterozygous samples, which illustrates the MNP markers of the developed respiratory syncytial virus Suitability of assays for detecting genetic variation within strain populations.
  • RNA of all strains or samples used to construct the respiratory syncytial virus MNP fingerprint database was extracted by conventional CTAB method and commercial kits, and the quality of RNA was detected by agarose gel and ultraviolet spectrophotometer. After sequence comparison of the sequencing data of the above six strains, the main genotype of each marker of each strain was obtained, and the MNP fingerprint of each strain was formed, which was entered into the database file to form the respiratory syncytial virus MNP fingerprint database.
  • the constructed MNP fingerprint database is based on the gene sequences of the detected strains and is therefore compatible with all high-throughput sequencing data. By comparing the MNP fingerprints of the strains obtained by each detection with the constructed MNP fingerprint database, the MNP fingerprint database constructed by the MNP fingerprints of the strains with differences in main genotypes realizes the co-construction and sharing of the database and update anytime.
  • Embodiment 6 application in fine typing of respiratory syncytial virus
  • the above six respiratory syncytial virus strains were detected by using the primer combination and MNP marker combination detection method, and the MNP fingerprints of each strain were obtained. After pairwise comparison of the DNA fingerprints of each strain, compare them with the published genome sequence of respiratory syncytial virus and the constructed fingerprint database. At least one MNP marker with a main genotype difference was defined as a new variant to achieve fine typing of respiratory syncytial virus.
  • the detection of 6 RSV samples is shown in Table 5. Among the 6 RSV samples detected, 1 and the other 5 have differences in the main genotypes of the 5 MNP markers, which may have mutated into different transform. Therefore, the resolution of RSV in the method reaches the level of single base, and fine typing of RSV in samples can be realized.

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Abstract

La présente invention concerne le domaine technique de la biologie moléculaire, et concerne une combinaison de marqueurs MNP, une combinaison de paires d'amorces et un kit de virus respiratoire syncytial, et leur utilisation. La combinaison de marqueurs MNP comprend 15 marqueurs, et les séquences nucléotidiques spécifiques sont représentées dans SEQ ID NO : 1 à SEQ ID NO : 15 ; la combinaison de paires d'amorces comprend 15 paires d'amorces, et les séquences d'amorces spécifiques sont représentées dans SEQ ID NO :16 à SEQ ID NO : 45. La combinaison de marqueurs MNP peut identifier spécifiquement le virus respiratoire syncytial et détecter finement les mutations ; les amorces n'interfèrent pas entre elles ; en intégrant la technologie d'amplification et de séquençage multiplex, l'analyse des séquences peut être effectuée en une seule fois sur tous les marqueurs de plusieurs échantillons.
PCT/CN2021/129169 2021-11-06 2021-11-06 Combinaison de marqueurs mnp, combinaison de paire d'amorces et kit de virus respiratoire syncytial et son utilisation WO2023077491A1 (fr)

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