WO2023077487A1 - Combinaison de marqueurs mnp d'adénovirus, combinaison de paires d'amorces, kit et utilisation associée - Google Patents

Combinaison de marqueurs mnp d'adénovirus, combinaison de paires d'amorces, kit et utilisation associée Download PDF

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WO2023077487A1
WO2023077487A1 PCT/CN2021/129165 CN2021129165W WO2023077487A1 WO 2023077487 A1 WO2023077487 A1 WO 2023077487A1 CN 2021129165 W CN2021129165 W CN 2021129165W WO 2023077487 A1 WO2023077487 A1 WO 2023077487A1
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adenovirus
mnp
marker
combination
detection
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陈利红
彭海
高利芬
周俊飞
李论
肖华峰
李甜甜
方治伟
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
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    • 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
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    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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 an adenovirus MNP marker combination, primer pair combination, kit and application thereof.
  • Adenovirus is a double-stranded DNA virus. Human adenoviruses are known to have 52 serotypes, named adl-ad52 respectively, and divided into six subgroups (A, B, C, D, E, and F). Adenovirus can infect the respiratory tract, gastrointestinal tract, urinary tract, bladder, eye, liver, etc. About 1/3 of the known serotypes of human adenovirus are usually associated with human diseases, but one serotype can cause different clinical diseases ; On the contrary, different serotypes can also cause the same disease. Adenovirus can infect a series of mammalian cells, with a wide host range and low pathogenicity to humans. The adenovirus vector system is widely used for the expression of human and non-human proteins, and is a commonly used research vector in laboratories. one.
  • the existing adenovirus typing system is mainly based on serotype, which has the defects of cross-reaction and low accuracy; the classic adenovirus detection method, including morphological examination based on isolation and culture, serological detection and antigen detection, consumes a lot of time in detection. There are one or more limitations in terms of time, operational complexity, detection throughput, and detection of variants.
  • the development of molecular biology makes it possible to identify and type by detecting the DNA of adenovirus. Therefore, the development of a rapid, accurate, and variable-monitoring adenovirus detection and analysis method is of great significance for the scientific research and application of adenoviruses.
  • DNA detection technologies such as PCR technology, whole genome, targeted molecular marker detection technology and metagenomic sequencing have been developed successively.
  • Targeted enrichment of target microorganisms avoids a large amount of data waste and background noise caused by metagenomic sequencing, and has the advantages of less sample requirements, accurate diagnostic results, saving data volume, and detecting low-frequency mutations.
  • the molecular markers detected by existing targeted detection technologies mainly include SNP and SSR markers.
  • SSR markers are recognized as the most polymorphic markers, but their number is small in microorganisms; SNP markers are huge in number, densely distributed, and are dimorphic markers, and the polymorphism of a single SNP marker is not enough to capture potential alleles in microbial populations genetic diversity. Therefore, the development of new molecular markers with high polymorphism and their detection technology has become an urgent technical problem to be solved.
  • 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 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 can realize the classification and traceability of multi-allelic genotypes in populations, and has application potential in the identification of adenoviruses, the construction of fingerprint databases, and the detection of genetic variation.
  • MNP marker and its detection technology MNP marker method can realize the classification and traceability of multi-allelic genotypes in populations, and has application potential in the identification of adenoviruses, the construction of fingerprint databases, and the detection of genetic variation.
  • MNP marker and its detection technology MNP marker method can realize the classification and traceability of multi-allelic genotypes in populations, and has application potential in the identification of adenoviruses, the construction of fingerprint databases, and the detection of genetic variation.
  • the present invention has developed MNP markers and detection primers for pathogenic microorganism adenoviruses, and the developed marker and primer combinations will be used to formulate national standards for pathogen detection (plan number 20201830-T-469), which will be released by the end of 2021. release.
  • the purpose of the embodiment of the present invention is to provide an adenovirus MNP marker combination, primer pair combination, kit and application thereof, which can identify and detect variation of adenovirus, and have multi-target, high-throughput, high-sensitivity and fine typing Effect.
  • the present invention adopts the following technical solutions:
  • an adenovirus MNP marker combination refers to the adenovirus genome that is screened to distinguish it from other species and has multiple nucleotide polymorphisms within the species Genomic regions of the genome, including 15 markers from MNP-1 to MNP-15 on the adenovirus reference sequence.
  • the specific nucleotide sequence of the marker combination of MNP-1 ⁇ MNP-15 is shown in 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 nucleotide sequence is as SEQ ID NO .16-shown in SEQ ID NO.45.
  • 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 adenovirus MNP marker includes the primer pair combination.
  • kit also includes a multiplex PCR master mix.
  • adenovirus MNP marker combination or the primer pair combination or the detection kit in the qualitative detection of adenovirus for non-diagnostic purposes, and the application in the preparation of adenovirus qualitative detection products.
  • the MNP marker of the adenovirus or the multiplex PCR primer pair combination or the detection kit is provided in the identification of adenovirus, the construction of a DNA fingerprint database, and the detection of genetic variation Applications.
  • the kit of the present invention to carry out the first round of multiplex PCR amplification to the total DNA and the blank control, and the number of cycles is not higher than 25;
  • the amplified product is purified, add sample tags and next-generation sequencing adapters based on the second round of PCR amplification; quantify the second-round amplified product after purification; detect multiple strains by combining the second-round amplified product High-throughput sequencing is performed after equal amounts are mixed; the sequencing results are compared to the reference sequence of the adenovirus, and the number of detected sequences and genotype data in the total DNA are obtained.
  • the quality control scheme and determination method use the DNA of adenovirus with known copy number as the detection sample, evaluate the sensitivity, accuracy and specificity of the kit for detecting adenovirus, and formulate the kit for detecting adenovirus.
  • the quality control scheme and determination method for viruses are used for adenovirus identification.
  • 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.
  • the genotype of each MNP marker of the strain to be compared Compare. If there are 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 adenovirus identified from the sample is entered into the database file to form the DNA fingerprint database of the adenovirus; each time a different sample is identified, passed Compared with the DNA fingerprint database of the adenovirus, identify whether the adenovirus in the sample and the strain in the database have a main genotype at the MNP marker (a genotype supported by more than 50% of the sequencing fragments at an MNP marker)
  • the adenovirus with main genotype difference in at least one MNP marker is a new variant type, which is included in the DNA fingerprint database.
  • adenovirus typing When used for adenovirus typing, it is to identify the adenovirus in the sample to be tested, and obtain the genotype of each MNP site; collect the genome sequence of the adenovirus published on the Internet and the constructed adenovirus DNA fingerprint database Constitute the adenovirus reference sequence library; compare the genotype of the adenovirus in the sample to be tested with the reference sequence library of the adenovirus, screen the genetically consistent or closest strains, and obtain the typing of the adenovirus in the sample to be tested . According to the comparison result with the reference sequence library, it is identified whether the adenovirus in the sample is an existing type or a new variant, so as to realize fine typing of the adenovirus.
  • the present invention has the following advantages:
  • the invention provides an adenovirus MNP marker, primer pair combination, kit and application thereof.
  • the provided 15 MNP markers of adenovirus 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
  • the demand for high-sensitivity detection meets the requirements for adenovirus standard and sharable fingerprint data construction; the demand for accurate detection of genetic variation between adenovirus strains; the need for identification of adenovirus homozygosity and heterozygosity, for the scientific research of adenovirus , Monitoring and prevention provide technical support.
  • the present invention is the first in the field of adenovirus, and there are no related literature reports.
  • Figure 1 is a schematic diagram of MNP marker polymorphism
  • Fig. 2 is the flow chart of screening and primer design of adenovirus MNP marker
  • Fig. 3 is the detection flowchart of MNP mark
  • MNP markers are mainly developed based on reference sequences. According to the resequencing data of reported adenovirus representative strains, large-scale MNP markers that are distinguished from other species, polymorphic within adenovirus species, and conserved on both sides can be mined; through MNP markers, two The conserved sequence on the side can be used to design MNP marker detection primers suitable for multiplex PCR amplification; and then according to the test results of the standard, a set of MNP markers with the largest polymorphism and high specificity and the best compatibility can be screened out. Primer combinations and detection kits.
  • Example 1 The screening of adenovirus 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.
  • Adenoviral DNA with known copy number was added to human genomic DNA to prepare a template of 1000 copies/reaction, which was detected by the MNP marker detection method, and four repeated sequencing libraries were constructed.
  • the detection of MNP markers in the medium screens the primer combinations with uniform amplification and optimal compatibility, and finally screens to obtain the detection primer combinations of 15 MNP markers described in the present invention, specifically as error! Reference source not found. shown.
  • Example 2 The performance evaluation and threshold setting of the MNP markers and primers for identifying adenoviruses
  • adenovirus DNA with a known copy number provided by the Hubei Provincial Center for Disease Control and Prevention was added to human genomic DNA to prepare 1 copy/reaction, 10 copy/reaction and 100 copy/reaction adenovirus mock samples .
  • an equal volume of sterile water was set as a blank control.
  • the kit can stably detect more than 7 MNP sites in samples with 10 copies/reaction, and detect at most 1 MNP site in a small number of samples with 0 copies/reaction.
  • the kit can clearly distinguish samples with 10 copies/reaction and 0 copies/reaction, and has technical stability and detection sensitivity as low as 10 copies/reaction.
  • the reproducibility and accuracy of the MNP marker detection method for detecting adenoviruses were evaluated. Specifically, the 12 sets of data of 100 copies of samples were compared in pairs, and the results are shown in Table 3. The number of MNP markers with differences in the main genotypes is all 0;
  • r represents the recurrence rate, that is, the main genotype can be reproduced
  • the sequence aligned to adenovirus can be detected in 1 copy/reaction sample, covering at least 1 MNP marker. In some blank controls, the sequence of adenovirus was also detected. 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 megabases. 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 megabases, one experiment for most samples can ensure that the sequencing of each marker is covered. The number of 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 adenovirus 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 adenovirus in the test sample.
  • the average value of the noise index of adenovirus in the blank control is 0.06%, while the average value of the signal index in the 1-copy sample is 0.41%, and the signal-to-noise ratio of the 1-copy sample and the blank control The average value of is 5.19. 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 10 copies of the sample and the blank control is 46.5, and in the 15 sets of data of 10 copies/reaction, at least 7 MNP markers can be stably detected, accounting for the total markers 46.7%. Therefore, in the case of ensuring accuracy, this standard stipulates that the signal-to-noise ratio threshold of adenovirus is 20, that is, when the signal-to-noise ratio of adenovirus in the sample is greater than 20, and the marker detection rate is greater than or equal to 30%, the sample is judged to be The nucleic acid of adenovirus was detected in the Therefore, the kit provided by the present invention can accurately and sensitively detect 10 copies/reaction of adenovirus.
  • kits and MNP marker combination detection method to detect 4 progeny strains of an adenovirus strain provided by the Hubei Provincial Center for Disease Control and Prevention, the samples were named S1-S4 in sequence, and each MNP marker The average sequencing coverage was 3103 times, and all 15 MNP markers could be detected for each strain (Table 5). Compare the fingerprints of the 4 strains in pairs, and the results are shown in Table 4 Wrong! Reference source not found. As shown, 1 (S-2) and 3 adenoviruses detected together with the same batch had major genotype differences of partial markers (Table 5), and there were variations 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 adenovirus strains in different laboratories, thereby ensuring the comparability of research results, which is important for the science of adenoviruses. Research matters. In clinical practice, the diagnostic scheme can be considered according to whether differential markers affect drug resistance.
  • the adenovirus group As a group organism, some individuals within the adenovirus group mutate, making the group no longer homozygous and forming a heterogeneous heterozygous group, which affects the stability and consistency of the microbial phenotype, especially in the test.
  • Such variants appear as alleles outside of the marker's major genotype when the population is tested for molecular markers. When the variant individual has not yet accumulated, it only accounts for a very small part of the population, showing a low frequency allele type. Low-frequency allele types are often mixed with technical errors, making them difficult to distinguish with existing techniques.
  • the present invention detects highly polymorphic MNP markers. The technical error rate of MNP markers is significantly lower than that of SNP markers, based on the fact that multiple errors are less likely to occur simultaneously than one error.
  • 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 calculated according to the following 8 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, 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 adenovirus MNP marker combination detection Applicability of the method for detecting genetic variation within strain populations.
  • the DNA of all strains or samples used to construct the adenovirus DNA fingerprint database was extracted by conventional CTAB method and commercial kits, and the quality of the DNA was detected by agarose gel and ultraviolet spectrophotometer. If the ratio of the absorbance value of the extracted DNA at 260nm to 230nm is greater than 2.0, the ratio of the absorbance value at 260nm to 280nm is between 1.6 and 1.8, the main band of DNA electrophoresis is obvious, and there is no obvious degradation and RNA residue, it means that the genomic DNA has reached Relevant quality requirements, follow-up experiments can be carried out.
  • 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 adenovirus DNA 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.
  • Embodiment 6 application in adenovirus fine typing
  • the above four adenovirus strains were detected by using the primer combination and MNP marker combination detection method, and the MNP fingerprints of each strain were obtained.
  • Construct a reference sequence library of adenovirus which consists of the published genome sequence of adenovirus and the MNP fingerprint database of the constructed adenovirus; compare the MNP fingerprint of each strain with the reference sequence library constructed, and screen to obtain and The strain with the closest genetic distance in the sequence library; if the genotype is 100% identical to the existing strain, it is an existing type, and if there is a main genotype difference at at least one MNP site, it is a new variant, realizing the adenovirus fine classification.
  • the detection of 4 adenovirus samples was wrong! Reference source not found.

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Abstract

La présente invention divulgue une combinaison de marqueurs MNP d'adénovirus, une combinaison de paires d'amorces, un kit et une utilisation associée. La combinaison de marqueurs MNP comprend 15 marqueurs, et la séquence nucléotidique de la combinaison de marqueurs MNP est représentée par les SEQ ID NO : 1 à SEQ ID NO : 15. La combinaison de paires d'amorces comprend 15 paires d'amorces, et les séquences nucléotidiques de celles-ci sont représentées par SEQ ID NO : 16 à SEQ ID NO : 45. La combinaison de marqueurs MNP permet d'identifier spécifiquement l'adénovirus et d'effectuer une typification fine. Les amorces n'interfèrent pas entre elles. Associée à de multiples technologies d'amplification et de séquençage, la combinaison de marqueurs MNP permet d'effectuer une analyse de séquence sur toutes les combinaisons de marqueurs dans plusieurs échantillons à la fois. La combinaison de marqueurs MNP possède les avantages suivants : haut débit, cibles multiples, haute spécificité, haute sensibilité et indépendance vis-à-vis de la culture. Elle peut être appliquée à l'identification et à la détection des variations génétiques de l'adénovirus dans de grands échantillons et est importante pour la recherche scientifique et la surveillance épidémiologique de l'adénovirus.
PCT/CN2021/129165 2021-11-06 2021-11-06 Combinaison de marqueurs mnp d'adénovirus, combinaison de paires d'amorces, kit et utilisation associée WO2023077487A1 (fr)

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CN112687344A (zh) * 2021-01-21 2021-04-20 予果生物科技（北京）有限公司 一种基于宏基因组的人腺病毒分子分型和溯源方法及系统
CN112813194A (zh) * 2019-11-18 2021-05-18 宁波海尔施基因科技有限公司 一种基于毛细管电泳的腺病毒分型检测试剂盒及其使用方法
CN113249519A (zh) * 2021-04-21 2021-08-13 深圳市儿童医院 一种用于外周血样本的腺病毒检测分型试剂盒及方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077260A1 (fr) * 1999-06-15 2000-12-21 Genomic Profiling Systems, Inc. Etablissement de profil genomique: procede rapide permettant d'analyser un echantillon biologique complexe afin de deceler la presence de nombreux types d'organismes
US20030186244A1 (en) * 2002-03-26 2003-10-02 Perlegen Sciences, Inc. Pharmaceutical and diagnostic business systems and methods
US20120264641A1 (en) * 2009-12-23 2012-10-18 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Methods and kits for identifying human adenovirus serotypes
CN105671212A (zh) * 2016-04-14 2016-06-15 中国人民解放军军事医学科学院放射与辐射医学研究所 腺病毒分型基因芯片的制备和用途
CN112813194A (zh) * 2019-11-18 2021-05-18 宁波海尔施基因科技有限公司 一种基于毛细管电泳的腺病毒分型检测试剂盒及其使用方法
CN112687344A (zh) * 2021-01-21 2021-04-20 予果生物科技（北京）有限公司 一种基于宏基因组的人腺病毒分子分型和溯源方法及系统
CN113249519A (zh) * 2021-04-21 2021-08-13 深圳市儿童医院 一种用于外周血样本的腺病毒检测分型试剂盒及方法

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