WO2023077484A1 - Combinaison de marqueurs mnp de cinq entérovirus humains, combinaison de paires d'amorces, kit et utilisations associées - Google Patents

Combinaison de marqueurs mnp de cinq entérovirus humains, combinaison de paires d'amorces, kit et utilisations associées Download PDF

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WO2023077484A1
WO2023077484A1 PCT/CN2021/129162 CN2021129162W WO2023077484A1 WO 2023077484 A1 WO2023077484 A1 WO 2023077484A1 CN 2021129162 W CN2021129162 W CN 2021129162W WO 2023077484 A1 WO2023077484 A1 WO 2023077484A1
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mnp
human
marker
combination
detection
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彭海
高利芬
周俊飞
李甜甜
陈利红
李论
方治伟
肖华峰
万人静
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江汉大学
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  • the embodiment of the present invention relates to the field of biotechnology, in particular to a combination of MNP markers of five human enteroviruses, a combination of primer pairs, a kit and applications thereof.
  • Human enterovirus is a genus of Picornaviridae, and its genetic material is RNA virus. It has been reported that poliovirus, Coxsackievirus (Coxsackievirus), enterocytopathic human orphan virus (ECHO, referred to as Echovirus) ) and new enteroviruses, etc., with a total of more than 70 serotypes. Humans are the only natural host of enteroviruses, and the virus spreads through close contact between people (through fingers, tableware and food). Feces - oral is the main route of transmission, and occasionally can also spread through droplets. Virus shedding from feces takes longer and can last for several weeks. Enteroviruses are infectious diseases caused by viruses.
  • the mild clinical manifestations are fatigue, fatigue, low-grade fever, etc., and severe cases can cause systemic infection, brain, spinal cord, heart, liver and other important organs are damaged, the prognosis is poor, and sequelae or cause death.
  • This type of disease is distributed all over the world and occurs throughout the year in the tropics and subtropics, and the incidence is high in warm, humid, poor sanitation and crowded areas.
  • Human enteroviruses are widely distributed and have many types.
  • the known coxsackieviruses are divided into two groups, a and b.
  • the EV71 type has attracted much attention because it caused the epidemic of hand, foot and mouth disease in children, and it is the object of key quarantine and vaccination.
  • Metagenome sequencing technology is another technology for detecting human enteroviruses, but it often includes a large amount of host sequencing data. When testing samples with low viral loads, ultra-deep sequencing is especially required, resulting in high costs. Therefore, the development of a rapid, accurate, one-time high-throughput human enterovirus detection and analysis method for the detection and typing of various human enteroviruses is of great significance for the detection and epidemic prevention of human enteroviruses.
  • 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 High-throughput, sensitive detection and precise typing of human enteroviruses, with less sample requirements, simultaneous coverage of coxsackievirus, echovirus, type C, type D and EV71 human enterovirus, accurate diagnostic results and saving data
  • the advantages of quantity and subtype detection are provided.
  • MNP markers refer to polymorphic markers caused by multiple nucleotides in an upper region of the genome. Compared with traditional 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) species discrimination Strong ability, only a small amount of MNP markers can realize species identification, reducing the detection error rate.
  • the MNP labeling method based on super 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 built 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 MNP marker method has application potential in the simultaneous identification and typing of multiple viruses, fingerprint database construction, and genetic variation detection.
  • MNP marker method has application potential in the simultaneous identification and typing of multiple viruses, fingerprint database construction, and genetic variation detection.
  • the combination of markers and primers developed in this invention will also be used to formulate national standards for pathogen detection (plan number 20201830-T-469), which will be released by the end of 2021.
  • the purpose of the present invention is to provide a MNP marker combination, primer pair combination, kit and application thereof of 5 kinds of human enteroviruses, which can treat Coxsackievirus, echovirus, C type, D type and EV71 type in total 5
  • the identification and mutation detection of human enteroviruses have the effects of multi-target, high throughput, high sensitivity, fine typing and isolation-free culture.
  • the present invention adopts the following technical solutions:
  • a MNP marker combination of 5 kinds of human enteroviruses said MNP marker combination refers to the conserved, differentiating from other species and possessing Genomic regions with multiple nucleotide polymorphisms, including 52 markers from MNP-1 to MNP-52 on the reference sequence of human enteroviruses.
  • Table 1 of the description further explains it.
  • the start and end positions of the MNP markers marked in Table 1 are determined based on the reference sequence corresponding to the same row of MNP in Table 1.
  • a combination of multiple PCR primer pairs for detecting the MNP marker combination includes 52 pairs of primers, and the specific primer nucleotide sequence is as SEQ ID NO .53-SEQ ID NO.156, wherein ID NO.53-SEQ ID NO.104 is the upper primer ID NO.105-SEQ ID NO.156 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 combination of the five human enterovirus MNP markers includes the combination of primer pairs.
  • kit also includes a multiplex PCR master mix.
  • the MNP marker combination of the five human enteroviruses or the multiple PCR primer pair combination or the detection kit is provided in the identification of human enteroviruses, genetic variation detection, MNP Construction of fingerprint database and application in fine typing.
  • 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
  • high-throughput sequencing is performed by mixing equal amounts of the second-round amplification products; the sequencing results are compared to the reference sequence of the human enterovirus, and the number of detected sequences and the number of sequences detected in the cDNA are obtained.
  • genotype data According to the number of human enterovirus sequencing sequences obtained in the cDNA and the blank control and the number of detected MNP markers, data quality control and data analysis are performed on the sequencing data of the cDNA to obtain the detected in the sample. The number of human enterovirus MNP markers, the number of sequencing sequences covering each of the MNP markers and the genotype data of the MNP markers.
  • the quality control scheme and the determination method use the RNA of human enterovirus with known copy number as the detection sample, evaluate the sensitivity, accuracy and specificity of the kit for detecting human enterovirus, and formulate the kit Quality control scheme and determination method for detection of human enterovirus.
  • the detection of genetic variation of human enteroviruses 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 52 part or all of the MNP markers of each strain to be compared. Through genotype comparison, it was analyzed whether there were differences in the main genotypes of the strains to be compared on the 52 common detected 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. As an alternative, it is also possible to amplify some or all of the 52 MNP markers of the strains to be compared by single-plex PCR according to the purpose of the research, and then perform Sanger sequencing on the amplified products. The genotypes of common MNP markers detected by the strains were compared for comparison.
  • 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 markers of the human enterovirus identified from the sample are entered into the database file to form the MNP fingerprint database of the human enterovirus respectively;
  • identify different samples for the second time by comparing with the MNP fingerprint database of the human enterovirus, identify whether the human enterovirus in the sample and the strain in the database have a main genotype in the MNP marker (in a MNP Markers have more than 50% of the differences in the genotypes supported by the sequencing fragments), and the human enteroviruses with major genotype differences in at least one MNP marker are new variant types, which are included in the MNP fingerprint database.
  • the human enterovirus When used for typing the human enterovirus, it is to identify the human enterovirus in the test sample, and obtain the genotype of each MNP site; collect the genome sequence of the human enterovirus disclosed on the Internet Compose the human enterovirus reference sequence library with the constructed human enterovirus MNP fingerprint database; compare the genotype of the human enterovirus in the sample to be tested with the reference sequence library of the human enterovirus, and screen The genetically identical or closest strain is used to obtain the typing of the human enterovirus in the sample to be tested. According to the comparison result with the reference sequence library, it is identified whether the human enterovirus in the sample is an existing type or a new variant, so as to realize fine typing of the human enterovirus.
  • This invention is the first in the field of human enteroviruses, and there are no related literature reports; MNP markers are mainly developed based on reference sequences, and large-scale identification of human enteroviruses from other species can be mined based on the resequencing data of nearly one million pieces of human enteroviruses that have been reported.
  • MNP markers that are polymorphic within human enterovirus subtypes and conserved sequences on both sides; design primers for MNP marker detection suitable for multiplex PCR amplification; and then screen out a set of polymorphisms with the largest and specificity through the test of positive samples
  • a set of MNP markers, multiplex PCR primer combinations and detection kits with the highest and best primer compatibility are used in the detection of human enteroviruses, the construction of MNP fingerprints, the detection of genetic variation and other related applications, for the detection of human enteroviruses, Scientific research and prevention and control provide technical support.
  • the present invention has the following advantages:
  • the invention provides MNP marker combination, primer pair combination, kit and application thereof for identifying and typing five kinds of human enteroviruses.
  • the provided 52 MNP markers of human enteroviruses and their primer combinations can be used for multiplex PCR amplification, combined with the next-generation sequencing platform to sequence the amplified products, meeting the requirements of high-throughput and high-speed Efficiency, high accuracy and high sensitivity detection and differentiation requirements; to meet the needs of accurate detection of human enterovirus genetic variation; to meet the needs of building a standard and shareable fingerprint database of human enterovirus for the monitoring of epidemic strains.
  • Figure 1 is a schematic diagram of MNP marker polymorphism
  • Fig. 2 is the screening and primer design flowchart of human enterovirus MNP marker
  • Fig. 3 is the detection flowchart of MNP mark
  • Example 1 The screening of human enterovirus MNP marker combination and the design of multiple PCR amplification primers
  • 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.
  • the primer combinations (Table 1) provided by the present invention have all detected the specific markers of each virus in 5 human enterovirus single samples tested, and in 3 repetitions of mixed samples, all mixed samples All human enterovirus types were also detected. Because of the diversification of the human enterovirus genome, not all the specific markers were detected, but the detected specific markers were all specifically detected in the expected virus samples, and could be distinguished from each other, and the specific virus could be identified. .
  • RNA of human enterovirus EV71 type, C type, D type, Coxsackie virus, and echovirus quantified by digital PCR provided by the Hubei Provincial Center for Disease Control and Prevention was reversed by a commercial kit. After recording into cDNA, they were added to human genomic DNA to prepare 1 copy/reaction, 10 copies/reaction and 100 copies/reaction of five human enterovirus simulation samples, and an equal volume of sterile water was set as a blank control. A total of 4 samples were detected for each virus, and 3 replicate libraries were constructed for each sample every day, and the detection was continued for 4 days, that is, 12 sets of sequencing data were obtained for each sample of each virus. Table 3 shows the data analysis of human enterovirus EV71 result.
  • the kit can detect 1 MNP site in a small number of samples with 0 copy/reaction, and can detect 2-4 MNP sites in a sample with 1 copy/reaction. More than 7 MNP sites were stably detected in samples with 10 copies/reaction, indicating that the detection sensitivity of the kit can be as low as 10 copies/reaction, and it has obvious technical stability when detecting samples with 10 copies/reaction and above sex.
  • the quality control plan is as follows:
  • the amount of sequencing data is greater than 16 megabases. The calculation is based on the fact that the number of MNP markers detected by each sample is at most 52, and the length of a sequencing fragment is 300 bases. Therefore, when the data volume is greater than 16 megabases, 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.
  • Blank control noise index P nc/Nc, wherein nc and Nc respectively represent the number of sequenced fragments and the total number of sequenced fragments of human enterovirus 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 human enterovirus in the test sample.
  • the average value of the noise index of the human enterovirus EV71 type in the blank control is 0.04%, while the average value of the signal index in the sample of 1 copy is 0.27%, and the average value of the sample of 1 copy and the blank control
  • the average value of the signal-to-noise ratio is 7.0. Therefore, the present invention stipulates that when the signal-to-noise ratio is greater than 10 times, it can be determined that the contamination in the detection system is acceptable.
  • the average value of the signal-to-noise ratio of human enterovirus EV71 in 10 copies of the sample and the blank control is 69.4, and in the 12 sets of data of 10 copies/reaction, at least 6 can be stably detected EV71-specific MNP markers accounted for 42.9% of the total markers. Therefore, in the case of ensuring accuracy and taking into account sensitivity, the criteria for judging the positiveness of human enterovirus in this patent is: when the signal-to-noise ratio of human enterovirus in the sample is greater than 34.7, and the marker detection rate is greater than or equal to 21.5%, it is determined that Nucleotides of human enterovirus were detected in the sample.
  • the average signal-to-noise ratios of human enterovirus type C, type D, coxsackie virus, and echovirus were 68.4, 69.2, 69.4, and 68; in 12 sets of data at 10 copies/reaction Among them, the specific markers of human enterovirus type C and type D, coxsackie virus and echovirus that can be stably detected accounted for 80.0%, 71.4%, 66.7% and 66.7% of the specific markers respectively. Therefore, in the guarantee In the case of both accuracy and sensitivity, the criteria for judging the positiveness of human enterovirus in this patent is that the signal-to-noise ratio is greater than 30%, and when the marker detection rate meets the following conditions:
  • the detection rate of the type C human enterovirus marker in the sample is greater than or equal to 40.0%, it is determined that the nucleic acid of the type C human enterovirus has been detected in the sample.
  • the marker detection rate of the echovirus in the sample is greater than or equal to 33.4%, it is determined that the nucleic acid of the echovirus has been detected in the sample.
  • the kit provided by the present invention can accurately and sensitively detect 5 kinds of human enteroviruses as low as 10 copies/reaction.
  • the RNAs of type influenza virus, type C influenza virus, avian influenza virus and Zika virus were mixed together to prepare a mixed template, and a blank template was used as a control to detect pathogens in the mixed template using the kit provided by the present invention.
  • repeated experiments As shown in Table 6, the MNP marker sites of the five human enteroviruses can be specifically detected in three repeated experiments.
  • the nucleic acids of 5 kinds of human enteroviruses were detected in all of them, indicating that the MNP marker and the high specificity of the kit for detecting target microorganisms in complex templates.
  • Embodiment 3 detection of genetic variation between human enterovirus strains
  • the kit was used to detect 4 backup strains of human enterovirus EV71 provided by the Hubei Provincial Center for Disease Control and Prevention.
  • the samples were named S1-S4 in turn, and the average sequencing coverage of each sample was 2325 times.
  • Each strain can detect 10 MNP markers (Table 6).
  • the fingerprints of the four strains were compared in pairs, and the results are shown in Table 7.
  • S-2 There is one (S-2) and three human enterovirus EV71 types detected together in the same batch, all of which have partially marked major genes Type difference (Table 6), there is variation among strains.
  • kits to identify genetic variation between strains by detecting MNP markers can be used to ensure the genetic consistency of the same named human enterovirus strains in different laboratories, thereby ensuring the comparability of research results. scientific research 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.
  • Table 8 The critical value for determining the suballelic genotype under partial sequencing depth
  • the nucleotides of the two strains with different genotypes are divided into the following 8 ratios: 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/100 , 7/100 mixed, artificial heterozygous samples were prepared, each sample was detected 3 times, and a total of 24 sequencing data were obtained.
  • markers of heterozygous genotypes were detected in 24 artificial heterozygous samples, which shows that the developed human enterovirus MNP marker detection Applicability of the method for detecting genetic variation within strain populations.
  • RNA of all strains or samples used to construct the human enterovirus 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.
  • MNP marker screening the genotypes of each virus at MNP markers were obtained, and these genotypes constituted the reference genotype library of MNP markers.
  • the kit provided by the present invention the five kinds of human enteroviruses shown in Table 2 are tested for MNP markers, and the sequence data obtained are compared with the reference genotype library to obtain the main genotype of each marker for each strain , form the MNP fingerprint of each virus strain, and input the database file, form the MNP fingerprint database of human enterovirus.
  • the constructed MNP fingerprint database can be continuously updated and enriched. Because the constructed database is based on the gene sequences of detected strains, it is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed, shared, and updateable at any time.
  • Embodiment 6 fine typing of human enterovirus
  • the nucleotides of human enterovirus were mixed together, and the blank sample was used as a control, and the kit provided by the present invention was used to detect the human enterovirus in the mixed template.
  • the results of 3 repeated experiments showed that the analysis was carried out according to the quality control scheme and determination scheme, the kit could detect type-specific MNP markers, and the identified MNP markers could be successfully identified according to the detected type-specific MNP markers.
  • type-specific MNP markers could only be detected specifically in the type, indicating that the kit has high specificity in detecting target microorganisms in mixed templates, and can be used for various types of human enteroviruses So it can be used for the monitoring of human enterovirus epidemic strains.

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Abstract

La présente invention appartient au domaine technique de la biologie moléculaire et divulgue une combinaison de marqueurs MNP de cinq entérovirus humains, une combinaison de paires d'amorces, un kit et des utilisations associées. La combinaison de marqueurs MNP comprend 52 marqueurs sur une séquence de référence génomique d'entérovirus humains, et les séquences nucléotidiques spécifiques sont telles que représentées dans SEQ ID NO :1 à SEQ ID NO : 52 ; et la combinaison de paires d'amorces possède les séquences nucléotidiques telles que représentées dans SEQ ID NO : 53 à SEQ ID NO : 156. La combinaison de marqueurs MNP peut identifier spécifiquement cinq entérovirus humains et distinguer finement les différents sous-types de chaque entérovirus humain.
PCT/CN2021/129162 2021-11-06 2021-11-06 Combinaison de marqueurs mnp de cinq entérovirus humains, combinaison de paires d'amorces, kit et utilisations associées WO2023077484A1 (fr)

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