WO2023077485A1 - 一种肺炎衣原体的mnp标记组合、引物对组合、试剂盒及其应用 - Google Patents

一种肺炎衣原体的mnp标记组合、引物对组合、试剂盒及其应用 Download PDF

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WO2023077485A1
WO2023077485A1 PCT/CN2021/129163 CN2021129163W WO2023077485A1 WO 2023077485 A1 WO2023077485 A1 WO 2023077485A1 CN 2021129163 W CN2021129163 W CN 2021129163W WO 2023077485 A1 WO2023077485 A1 WO 2023077485A1
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mnp
chlamydia pneumoniae
combination
marker
detection
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French (fr)
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李甜甜
彭海
高利芬
李论
肖华峰
方治伟
陈利红
周俊飞
万人静
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江汉大学
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the embodiment of the present invention relates to the field of biotechnology, in particular to a combination of MNP markers of Chlamydia pneumoniae, a combination of primer pairs, a kit and applications thereof.
  • Chlamydia pneumoniae (Chlamydia.pneumoniae) is an obligate intracellular parasitic Gram-negative small coccus, an important pathogen of human respiratory diseases, mainly transmitted through respiratory droplets. After entering the human body, endotoxin is produced, which can cause upper respiratory tract infections, such as sinusitis, otitis media and pharyngitis, and lower respiratory tract infections, such as bronchitis and pneumonia. Surveys in recent years have shown that 5% to 10% of acquired pneumonia, bronchitis and sinusitis are caused by Chlamydia pneumoniae. Human Chlamydia pneumoniae infection is prevalent worldwide, regardless of region, race and age.
  • Chlamydia pneumoniae The elderly, infirm and immunocompromised are more likely to be infected with Chlamydia pneumoniae, and it is easy to recur and difficult to control. Therefore, accurate and sensitive detection of Chlamydia pneumoniae is of great significance for early diagnosis and control of the disease.
  • Chlamydia pneumoniae is also a commonly used model pathogenic microorganism for laboratory research.
  • As a group creature in the interaction with the host and the environment, the individuals in the group will mutate.
  • this imperceptible variation will cause different laboratories or different periods of the same laboratory with the same named strains to be actually different, resulting in non-reproducible and non-comparable experimental results.
  • the heterogeneity of human Hella cell laboratories has led to a large number of incomparable experimental results and data waste. Therefore, the development of a rapid, accurate, and variable-monitoring Chlamydia pneumoniae detection and analysis method is of great significance for the scientific research and application of Chlamydia pneumoniae.
  • Classical detection methods for Chlamydia pneumoniae including isolation and culture, PCR technology, whole genome and metagenomic sequencing, etc., have one or more limitations in terms of time length, operational complexity, detection throughput, accuracy and sensitivity of detection of mutations, and cost.
  • the targeted molecular marker detection technology that combines ultra-multiplex PCR amplification and high-throughput sequencing can enrich target microorganisms in samples with low microbial content, avoiding a large amount of data waste and background caused by whole genome and metagenomic sequencing Noise, which has the advantages of less sample requirement, accurate diagnosis results, saving data volume, and detecting low-frequency variation.
  • 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 and detection primers for the pathogenic microorganism Bacillus subtilis The combination of markers and primers developed in the present invention will 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 embodiment of the present invention is to provide a Chlamydia pneumoniae MNP marker combination, primer pair combination, kit and application thereof, which can be used for qualitative and quantitative identification and variation detection of Chlamydia pneumoniae, with multi-target, high-throughput, high sensitivity and fine-grained effect.
  • the present invention adopts the following technical solutions:
  • a MNP marker combination of Chlamydia pneumoniae refers to the screening on the Chlamydia pneumoniae genome that is different from other species and has multiple nucleotide polymorphisms within the species Genomic regions of the genome, including 15 markers of MNP-1 to MNP-15 with AE001363 as the reference genome, and the specific nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.15.
  • Table 1 of the instruction manual further explains the combination of MNP-1 to MNP-15 markers.
  • 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. of.
  • the multiple PCR primer pair combination includes 15 pairs of primers, and the specific primer nucleotide sequence is as SEQ ID NO .16-SEQ ID NO.45, wherein ID NO.16-SEQ ID NO.30 is the upper primer, and ID NO.31-SEQ ID NO.45 is the lower primer.
  • each MNP-labeled primer includes an upper primer and a lower primer, which is further described in Table 1 of the instruction manual.
  • a detection kit for detecting the Chlamydia pneumoniae core MNP marker combination includes the primer pair combination.
  • kit also includes a multiplex PCR master mix.
  • 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, etc.
  • High-throughput sequencing was carried out after the amounts were mixed; the sequencing results were compared to the reference sequence of Chlamydia pneumoniae, and the number of detected sequences and genotype data in the total DNA were obtained.
  • the quality control scheme and the determination method use the DNA of Chlamydia pneumoniae whose copy number is known as the detection sample, evaluate the sensitivity, accuracy and specificity of the kit for detecting Chlamydia pneumoniae, and formulate the kit for detecting pneumoniae The quality control scheme and determination method for chlamydia.
  • 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. As an alternative, it is also possible to amplify the 15 markers of the strain to be compared by single-plex PCR, and then perform Sanger sequencing on the amplified products.
  • the sequence After obtaining the sequence, compare the genotype of each MNP marker of the strain to be compared. right. If there are MNP markers with inconsistent main genotypes, there is variation among the strains to be compared. When detecting the genetic variation within the strain, it is judged by statistical model whether the MNP markers in the strain to be tested detect sub-genotypes other than the main genotype. If the test strain has a subgenotype in at least one MNP marker, it is determined that there is genetic variation within the test strain.
  • the genotype data of the MNP marker of the Chlamydia pneumoniae identified from the sample will be entered into the database file to constitute the DNA fingerprint database of Chlamydia pneumoniae; when different samples are identified each time, by Compared with the DNA fingerprint database of Chlamydia pneumoniae, identify whether the Chlamydia pneumoniae in the sample and the strain in the database have a main genotype (a genotype supported by more than 50% sequencing fragments at a MNP marker) in the MNP marker Differences, Chlamydia pneumoniae with major genotype differences in at least one MNP marker is a new variation type, which is included in the DNA fingerprint database.
  • Chlamydia pneumoniae When used for typing Chlamydia pneumoniae, it is to identify the Chlamydia pneumoniae in the sample to be tested, and obtain the genotype of each MNP site; collect the genome sequence of Chlamydia pneumoniae published online and the constructed DNA fingerprint database of Chlamydia pneumoniae Compose a Chlamydia pneumoniae reference sequence library; compare the genotype of Chlamydia pneumoniae in the sample to be tested with the reference sequence library of Chlamydia pneumoniae, screen genetically consistent or closest strains, and obtain the type of Chlamydia pneumoniae in the test sample . According to the comparison result with the reference sequence library, it is identified whether the Chlamydia pneumoniae in the sample is an existing type or a new variant, so as to realize fine typing of the Chlamydia pneumoniae.
  • the present invention is the first in the field of Chlamydia pneumoniae, and there is no relevant literature report; MNP markers are mainly developed based on reference sequences. According to the resequencing data of the reported representative races of Chlamydia pneumoniae, large-scale identification of Chlamydia pneumoniae species, which are different from other species, can be mined. MNP markers with polymorphic and conserved sequences on both sides of the species; MNP marker detection primers suitable for multiplex PCR amplification can be designed through the conserved sequences on both sides of the MNP markers; and a set of multiple A set of MNP markers with the greatest morphism and high specificity, the most compatible primer combination and detection kit.
  • the invention provides an MNP marker combination, primer pair combination, kit and application thereof for identifying and typing Chlamydia pneumoniae.
  • the provided 15 MNP markers of Chlamydia pneumoniae 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 high-throughput, high-efficiency, high-accuracy and
  • the demand for high-sensitivity detection meets the requirements of Chlamydia pneumoniae standard and shareable fingerprint data construction; the demand for accurate detection of genetic variation among strains of Chlamydia pneumoniae; the demand for identification of homozygous and heterozygous Monitoring, database construction and scientific research provide technical support.
  • Figure 1 is a schematic diagram of MNP marker polymorphism
  • Fig. 2 is the screening and primer design flowchart of Chlamydia pneumoniae MNP marker
  • Fig. 3 is the detection flowchart of MNP mark
  • MNP markers suitable for detecting population organisms as detection targets.
  • MNP markers refer to polymorphic markers caused by multiple nucleotides in an upper region of the genome.
  • SSR markers and SNP markers MNP markers have the following advantages: (1) Alleles are rich, there are 2 n alleles on a single MNP site, which is higher than SSR and SNP markers, and it is suitable for the typical population of microorganisms Biological detection; (2) strong species discrimination ability, only a small amount of MNP markers can realize species identification, reducing the detection error rate.
  • the MNP labeling method for detecting MNP markers combines super multiplex PCR and next-generation high-throughput sequencing technology, which has the following advantages: (1) The output is base sequences, 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 sites in hundreds of thousands of samples at one time; (3) High sensitivity, using multiplex PCR to detect multiple targets at one time, Avoid high false negatives and low sensitivity caused by single target amplification failure; (4) High accuracy, use the second-generation high-throughput sequencer to sequence the amplification 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 development, screening and application of MNP markers have a good application basis in plants.
  • the present invention has developed the MNP marker site of Chlamydia pneumoniae, and the MNP marker combination refers to the genomic region screened on the Chlamydia pneumoniae genome that is distinguished from other species and has multiple nucleotide polymorphisms within the species, including Taking AE001363 as the 15 markers of MNP-1 to MNP-15 of the reference genome, the specific nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.15.
  • the present invention has developed the multiple PCR primer composition that is used to detect described Chlamydia pneumoniae MNP marker site, and described multiple PCR primer composition comprises 15 pairs of primers, and the nucleotide sequence of described 15 pairs of primers is as SEQ ID NO .16 ⁇ shown in SEQ ID NO.45.
  • the primers do not conflict with each other and can be efficiently amplified by multiplex PCR;
  • the multiplex PCR primer composition can be used in a detection kit for detecting the MNP marker site of Chlamydia pneumoniae.
  • the kit provided by the invention can accurately and sensitively detect 10 copies/reaction of Chlamydia pneumoniae.
  • the MNP marker of the present invention and the kit have high specificity for detecting Chlamydia pneumoniae in complex templates.
  • Chlamydia pneumoniae MNP marker combination, primer composition, kit and application of the present application will be described in detail below in combination with examples, comparative examples and experimental data.
  • Example 1 The screening of Chlamydia pneumoniae MNP marker combination and the design of multiple PCR amplification primers
  • MNP markers were obtained through sequence alignment.
  • 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 method for detecting the MNP markers is to amplify all the MNP markers at one time through multiplex PCR, sequence the amplified products through second-generation high-throughput sequencing, analyze the sequencing data, and evaluate the primers according to the detected markers. Combination compatibility.
  • Chlamydia pneumoniae DNA with known copy number provided by Hubei Provincial Center for Disease Control and Prevention was added to human genomic DNA to prepare a template with 1000 copies/reaction. Detect by the described MNP marker detection method, construct 4 repeated sequencing libraries, screen the designed primer combinations according to the detection results, and finally screen to obtain the four libraries provided by the present invention that can be detected and compatible. The best 15 MNP-labeled primer pair combinations are shown in Table 1.
  • Chlamydia pneumoniae nucleic acid standard with known copy number was added to human genomic DNA to prepare 1 copy/reaction, 10 copies/reaction and 100 copies/reaction Chlamydia pneumoniae mock samples.
  • an equal volume of sterile water was set as a blank control.
  • the detection process of MNP markers is shown in FIG. 3 .
  • 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 kit can accurately and sensitively detect Chlamydia pneumoniae with less than 10 copies/reaction.
  • the sequence aligned to Chlamydia pneumoniae can be detected in 1 copy/reaction sample, covering at least 1 MNP marker.
  • the sequence of Chlamydia pneumoniae 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 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 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.
  • 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 Chlamydia pneumoniae 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 Chlamydia pneumoniae in the test sample.
  • the mean value of the noise index of Chlamydia pneumoniae in the blank control is 0.03%, while the mean value of the signal index in the sample of 1 copy is 0.06%, the signal-to-noise ratio of the sample of 1 copy and the blank control
  • the average value of is 2.1, therefore, the present 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.
  • the average value of the signal-to-noise ratio of 10 copies of the sample and the blank control is 84, and in the 12 sets of data of 10 copies/reaction, at least 8 MNP markers can be stably detected, accounting for the total markers 53.3%. Therefore, in the case of ensuring accuracy, this standard stipulates that the signal-to-noise ratio threshold of Chlamydia pneumoniae is 40, that is, when the signal-to-noise ratio of Chlamydia pneumoniae in the sample is greater than 40, and the marker detection rate is greater than or equal to 30%, the sample is judged to be The nucleic acid of Chlamydia pneumoniae was detected. Therefore, the kit provided by the present invention can sensitively detect 10 copies/reaction of Chlamydia pneumoniae.
  • kits to identify the genetic variation between strains by detecting MNP markers can be used to ensure the genetic consistency of the same named Chlamydia pneumoniae strains in different laboratories, thereby ensuring the comparability of research results, which is of great significance to the scientific research of Chlamydia pneumoniae 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 S-1 are mixed into S according to the following 8 ratios:
  • In -2 nucleotides artificial heterozygous samples were prepared, each sample was detected 3 times, and a total of 24 sequencing data were obtained.
  • the ratio is as low as 1/1000, illustrating the applicability of the developed MNP marker detection method for Chlamydia pneumoniae in detecting genetic variation within strain populations.
  • the DNA of all strains or samples used to construct the Chlamydia pneumoniae 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 to form the MNP fingerprint of each strain.
  • the MNP fingerprints of samples with differences were entered into the constructed MNP fingerprint database.
  • the constructed MNP fingerprint database is based on the gene sequence of the detected strains, so 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 the application in fine typing of Chlamydia pneumoniae
  • a reference sequence library of Chlamydia pneumoniae is constructed, which consists of the published genome sequence of Chlamydia pneumoniae and the constructed DNA fingerprint database of Chlamydia pneumoniae; using the primer combination and MNP marker site detection method described in Example 2, each The MNP fingerprint of Chlamydia pneumoniae in the sample to be tested; the DNA fingerprint of each strain is compared with the reference sequence library constructed, and the strain with the closest genetic distance to the sequence library is obtained by screening; and the genotype of the existing strain 100 % the same, it is an existing variant, and there is a main genotype difference in at least one MNP site, it is a new variant, and the fine typing of Chlamydia pneumoniae is realized.

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Abstract

本发明公开了一种肺炎衣原体的MNP标记组合、引物对组合、试剂盒及其应用，所述MNP标记组合包括以AE001363为参考基因组的MNP-1～MNP-15的15个标记位点，具体的核苷酸序列如SEQ ID NO.1-SEQ ID NO.15所示；所述引物对组合包括15对引物，具体核苷酸序列如SEQ ID NO.16-SEQ ID NO.45所示。所述MNP标记组合可用于肺炎衣原体的鉴定和遗传变异监测。

Description

一种肺炎衣原体的MNP标记组合、引物对组合、试剂盒及其应用 技术领域
本发明实施例涉及生物技术领域，特别涉及一种肺炎衣原体的MNP标记组合、引物对组合、试剂盒及其应用。
背景技术
肺炎衣原体(Chlamydia.pneumoniae)是一种专性细胞内寄生的革兰氏阴性小球菌，是人类呼吸道疾病的重要病原体，主要通过呼吸道飞沫传播。进入人体后，产生内毒素，可引起上呼吸道感染，如鼻窦炎、中耳炎和咽炎，也可引起下呼吸道感染，如支气管炎和肺炎。近年来的调查显示，5％～10％的获得性肺炎、支气管炎和鼻窦炎是由肺炎衣原体引起。人类肺炎衣原体感染呈世界性流行，不分地域、不受种族和年龄限制，年老体弱者和免疫力低下者较易感染肺炎衣原体，且易反复发作，不易控制。因此，准确灵敏的肺炎衣原体的检测对于疾病早期诊断及控制具有重要意义。
另外，肺炎衣原体也是实验室进行研究常用的模式病原微生物。其作为群体生物，在和宿主、环境的互作中，群体内个体会发生变异。对于实验室的研究来说，这种不易被察觉的变异会导致不同实验室或同一实验室不同时期相同命名的菌株实际上并不相同，导致实验结果的不可重现和不可比较。人Hella细胞实验室间的异质性已经导致大量的实验结果的不可比较和数据浪费。因此，开发快速、准确的、可监测变异的肺炎衣原体检测分析方法对于肺炎衣原体的科学研究和应用都具有重要意义。
经典的肺炎衣原体检测方法，包括分离培养、PCR技术、全基因组和宏基因组测序等，在时长、操作复杂度、检测通量、检测变异的准确性和灵敏度、成本等方面存在一个或多个局限。融合超多重PCR扩增和高通量测序的靶向分子标记检测技术，可以在低微生物含量的样本中靶向的富集目标微生物，避免了全基因组和宏基因组测序带来的大量数据浪费和背景噪音，具有样本需要量少、诊断结果精确，节约数据量、检测低频变异的优势。
现有的靶向检测技术检测的分子标记主要包括SNP和SSR标记。SSR标记是公认的多态性最高的标记，但在微生物中数量少；SNP标记数量巨大，分布密集，是二态性标记，单个SNP标记的多态性不足以捕获微生物种群中潜在的等位基因多样性。因此，开发高多态性的新型分子标记及其检测技术，成为亟待解决的技术问题。
因此，亟需开发病原微生物枯草芽孢杆菌的MNP标记和检测引物。本发明所开发的标记和引物组合将用于制定病原体检测的国家标准(计划编号20201830-T-469)，该国家标准 将于2021年底发布。
发明内容
本发明实施例目的是提供一种肺炎衣原体的MNP标记组合、引物对组合、试剂盒及其应用，可以对肺炎衣原体进行定性定量的鉴定和变异检测，具有多靶标、高通量、高灵敏和精细分型的效果。
为实现上述目的，本发明采用如下技术方案：
在本发明的第一方面，提供了一种肺炎衣原体的MNP标记组合，所述MNP标记组合是指在肺炎衣原体基因组上筛选的区分于其他物种且在物种内部具有多个核苷酸多态性的基因组区域，包括以AE001363为参考基因组的MNP-1～MNP-15的15个标记，具体的核苷酸序列如SEQ ID NO.1-SEQ ID NO.15所示。
上述技术方案中，说明书表1对MNP-1～MNP-15的标记组合进一步说明，表1中标注的所述MNP标记的起始和终止位置是基于表1中MNP同一行对应的参考序列确定的。
在本发明的第二方面，提供了一种用于检测所述MNP标记组合的多重PCR引物对组合，所述多重PCR引物对组合包括15对引物，具体的引物核苷酸序列如SEQ ID NO.16-SEQ ID NO.45所示，其中ID NO.16-SEQ ID NO.30为上引物，ID NO.31-SEQ ID NO.45为下引物。
上述技术方案中，每个MNP标记的引物包括上引物和下引物，说明书表1进一步进行了说明。
在本发明的第三方面，提供了一种用于检测所述肺炎衣原体核心MNP标记组合的检测试剂盒，所述试剂盒包括所述的引物对组合。
进一步地，所述试剂盒还包括多重PCR预混液。
以及所述的肺炎衣原体的MNP标记组合、引物对组合、检测试剂盒在非诊断目的的肺炎衣原体定性检测中的应用，在制备肺炎衣原体定性检测产品中的应用。
在本发明的第四方面，提供了所述的肺炎衣原体的MNP标记组合或者所述的多重PCR引物对组合或者所述的检测试剂盒在肺炎衣原体的鉴定、DNA指纹数据库的构建、遗传变异检测中的应用。
以上所述的应用中，首先是获取待测样本的细菌总DNA；利用本发明的试剂盒对所述总DNA和空白对照进行第一轮多重PCR扩增，循环数不高于25个；对扩增产物进行纯化后，进行基于第二轮PCR扩增的样本标签和二代测序接头添加；对第二轮扩增产物纯化后定量；检测多个菌株时通过将第二轮扩增产物等量混合后进行高通量测序；测序结果比对到所述 的肺炎衣原体的参考序列上，获取在所述总DNA的检测序列数目和基因型数据。根据在所述总DNA和所述空白对照获得的肺炎衣原体测序序列数量和检出MNP标记的数目，对所述总DNA的测序数据进行数据质量控制和数据分析，获得检出MNP标记数目、覆盖每个所述MNP标记的测序序列数目和所述MNP标记基因型数据。
当用于肺炎衣原体鉴定时，根据在待测样品和空白对照中检出的肺炎衣原体的测序序列数量和检出MNP位点的数目，进行质控后判定待测样品中是否含有肺炎衣原体的核酸。其中，所述的质控方案和判定方法是以拷贝数已知的肺炎衣原体的DNA为检测样本，评估所述试剂盒检测肺炎衣原体的灵敏度、准确性和特异性，制定所述试剂盒检测肺炎衣原体时的质控方案和判定方法。
当用于肺炎衣原体遗传变异检测时，包括菌株间和菌株内部的遗传变异检测。菌株间的遗传变异检测包括利用所述的试剂盒和方法，获得待比较菌株各自在15个MNP标记的基因型数据。通过基因型比对，分析待比较菌株在所述15个MNP标记上的主基因型是否存在差异。若待比较菌株在至少一个MNP标记的主基因型存在变异，则判定两者存在遗传变异。作为一种备选方案，也可以通过单重PCR对待比较菌株的15个标记分别进行扩增，然后对扩增产物进行Sanger测序，获得序列后，对待比较菌株每个MNP标记的基因型进行比对。如果存在主基因型不一致的MNP标记，则待比较菌株之间存在变异。当检测菌株内部的遗传变异时，则通过统计模型判定在待测菌株所述的MNP标记是否检出主基因型以外的次基因型。若待测菌株在至少一个MNP标记存在次基因型，则判定待测菌株内部存在遗传变异。
当用于构建肺炎衣原体DNA指纹数据库时，将从样本中鉴定的肺炎衣原体的所述MNP标记的基因型数据，录入数据库文件，构成肺炎衣原体的DNA指纹数据库；每次鉴定不同的样本时，通过和所述肺炎衣原体的DNA指纹数据库比对，鉴定样本中的肺炎衣原体是否和数据库中的菌株在所述MNP标记存在主基因型(在一个MNP标记具有超过50％测序片段支持的基因型)的差异，在至少1个MNP标记存在主基因型差异的肺炎衣原体即为新的变异类型，收录进DNA指纹数据库。
当用于肺炎衣原体分型时，是对待测样本中的肺炎衣原体进行鉴定，获得每个所述MNP位点的基因型；收集网上公开的肺炎衣原体的基因组序列和已构建的肺炎衣原体DNA指纹数据库组成肺炎衣原体参考序列库；将待测样本中肺炎衣原体的基因型和所述肺炎衣原体的参考序列库进行比对，筛选遗传上一致或最接近的菌株，获得待测样本中肺炎衣原体的分型。根据同所述参考序列库的比对结果，鉴定样品中的肺炎衣原体是已有的型还是新的变型，实现对肺炎衣原体的精细分型。
本发明在肺炎衣原体领域属于首创，并未见相关文献报道；MNP标记主要基于参考序列开发，根据已报道的肺炎衣原体代表小种的重测序数据可以挖掘大规模的区分于其他物种、在肺炎衣原体物种内部多态、两侧序列保守的MNP标记；通过MNP标记两侧的保守序列可以设计适用于于多重PCR扩增的MNP标记检测引物；再根据标准品的测试结果，可筛选出一套多态性最大、特异性高的一套MNP标记、兼容性最好的引物组合和检测试剂盒。
本发明实施例中的一个或多个技术方案，至少具有如下技术效果或优点：
本发明提供了一种用于鉴定分型肺炎衣原体的MNP标记组合、引物对组合、试剂盒及其应用。所提供的肺炎衣原体的15个MNP标记和其引物组合，可进行多重PCR扩增，融合二代测序平台进行扩增产物的测序，满足对肺炎衣原体进行高通量、高效率、高准确性和高灵敏度检测的需求，满足肺炎衣原体标准的、可共享的指纹数据构建的要求；准确检测肺炎衣原体菌株间遗传变异的需求；鉴定肺炎衣原体纯合和杂合的需求，为肺炎衣原体的检测、变异监测、数据库构建和科学研究提供技术支撑。
附图说明
图1为MNP标记多态性原理图；
图2为肺炎衣原体MNP标记的筛选和引物设计流程图；
图3为MNP标记的检测流程图；
具体实施方式
为了便于理解本发明，下面结合附图和具体实施例，对本发明进行更详细的说明。附图中给出了本发明的较佳的实施例。但是，本发明可以以许多不同的形式来实现，并不限于本说明书所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
需要说明的是，除非另有定义，本说明书所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是用于限制本发明。
除非另有特别说明，本发明实施例中用到的各种原材料、试剂、仪器和设备等，均可通过市场购买得到或者可通过现有方法制备得到。
为解决上述技术问题，本申请的技术方案总体思路如下：
筛选适用于检测群体生物的MNP标记作为检测目标。MNP标记是指在基因组上一段区域内由多个核苷酸引起的多态性标记。与SSR标记和SNP标记相比，MNP标记具有以下优势：(1)等位基因丰富，单个MNP位点上有2 n种等位基因，高于SSR和SNP，适用于微生物这种典型的群体生物的检测；(2)物种区分能力强，只需要少量的MNP标记就能实现物 种鉴定，减少了检测错误率。检测MNP标记的MNP标记法融合超多重PCR和二代高通量测序技术，具有以下优势：(1)输出的是碱基序列，无需平行实验，可构建标准化的数据库进行共享共享；(2)高效率，利用样品DNA条形码，突破测序样品数量的局限，可一次性对成百上千份样本的数万个MNP位点分型；(3)高灵敏度，利用多重PCR一次检测多个靶标，避免单个靶标扩增失败导致高的假阴性和低的灵敏度；(4)高准确性，利用二代高通量测序仪对扩增产物测序数百次。
鉴于以上优点和特性，MNP标记及其检测技术MNP标记法可实现群体生物多等位基因型的分类与溯源，在病原微生物的鉴定、指纹数据库构建、遗传变异检测等方面都具有应用潜力。目前在微生物中，尚未有关于MNP标记的报道，也缺乏相应的技术。MNP标记法的开发、筛选和应用在植物中具有较好的应用基础。
因此，本发明开发了肺炎衣原体的MNP标记位点，所述MNP标记组合是指在肺炎衣原体基因组上筛选的区分于其他物种且在物种内部具有多个核苷酸多态性的基因组区域，包括以AE001363为参考基因组的MNP-1～MNP-15的15个标记，具体的核苷酸序列如SEQ ID NO.1-SEQ ID NO.15所示。
接着，本发明开发了用于检测所述肺炎衣原体MNP标记位点的多重PCR引物组合物，所述多重PCR引物组合物包括15对引物，所述15对引物的核苷酸序列如SEQ ID NO.16～SEQ ID NO.45所示。所述引物互相间不冲突，可以通过多重PCR进行高效的扩增；
所述多重PCR引物组合物可以用于检测肺炎衣原体MNP标记位点的检测试剂盒。
本发明所提供的试剂盒能准确灵敏的检测到10拷贝/反应的肺炎衣原体。
本发明的重现性试验中每个样品不同文库间、不同建库批次间MNP标记主基因型的差异对数为0，重现率r＝100％，准确率a＝100％。
本发明的MNP标记和所述试剂盒在复杂模板中检测肺炎衣原体具有高特异性。
下面将结合实施例、对比例及实验数据对本申请的一种肺炎衣原体的MNP标记组合、引物组合物、试剂盒及其应用进行详细说明。
实施例1肺炎衣原体MNP标记组合的筛选和多重PCR扩增引物的设计
S1、肺炎衣原体MNP标记组合的筛选
基于网上公开的12个肺炎衣原体不同分离株的基因组完整或部分序列，通过序列比对，获得15个MNP标记。对于网上不存在基因组数据的物种，也可以通过高通量测序获得待检测微生物物种代表小种的基因组序列信息，其中高通量测序可以是全基因组或简化基因组测序。为了保证所筛选标记的多态性，一般使用至少10个遗传上具有代表性的分离株的基因组序列作为参考。
筛选的15个MNP标记如表1所示：
表1所述MNP标记以及检测引物在参考序列上的起始位置
Figure PCTCN2021129163-appb-000001
所述步骤S1具体包括：
选择所述肺炎衣原体的一个代表株的基因组序列作为参考基因组，将所述基因组序列和所述参考基因组进行序列比对，获得所述肺炎衣原体各菌株的单核苷酸多态性标记；
在所述参考基因组上，以100-300bp为窗口，以1bp为步长进行窗口平移，筛选获得多个候选MNP标记区域，其中，所述候选MNP标记区域含有≥2个所述单核苷酸变异标记，且两端各30bp的序列上均不存在所述单核苷酸多态性标记；
在所述候选多核苷酸多态性标记区域中筛选区分度DP≥0.2的区域作为MNP标记；其中，DP＝d/t，t是在所述候选多核苷酸多态性标记区域中所有小种两两比较时的比较对数，d是在所述候选多核苷酸多态性标记区域中至少两个单核苷酸多态性差异的样品对数。
作为一种可选的实施方式，在所述参考基因组上，以100-300bp为窗口进行筛选时，也可选用其他步长，本实施方式采用步长为1bp，有利于全面的筛选。
S2、多重PCR扩增引物的设计
通过引物设计软件设计所述MNP标记的多重PCR扩增引物，引物设计遵循引物间互不干扰，所有引物可以组合成引物池进行多重PCR扩增，即所有设计的引物可以在一个扩增反应中均正常扩增。
S3、引物组合的检测效率评估
所述MNP标记的检测方法是通过多重PCR对所有MNP标记一次性进行扩增，通过二代高通量测序对扩增产物进行测序，对测序数据进行分析，根据检出的标记评价所述引物组合的兼容性。
使用湖北省疾控预防控制中心提供的拷贝数已知的肺炎衣原体DNA，加入到人基因组 DNA中，制备成1000拷贝/反应的模板。通过所述的MNP标记检测方法进行检测，构建4个重复的测序文库，根据检测结果对所设计的引物组合进行筛选，最终筛选获得本发明提供的在4个文库中都能检出、兼容性最好的15个MNP标记的引物对组合，具体如表1所示。
实施例2所述MNP标记和引物鉴定肺炎衣原体的阈值设置和性能评估
本实施例中，将拷贝数已知的肺炎衣原体核酸标准品加入到人基因组DNA中，制备1拷贝/反应、10拷贝/反应和100拷贝/反应的肺炎衣原体模拟样本。同时设置的等体积的无菌水作为空白对照。共计4个样本，每个样本每天构建3个重复文库，连续检测4天，即每个样本获得15组测序数据，具体如错误！未找到引用源。所示。根据在15次重复实验中，在空白对照和模拟样本中检出的肺炎衣原体MNP标记的测序片段数和标记数，评估检测方法的重现性、准确性、灵敏度，制定质控体系污染和目标病原体检出的阈值。
MNP标记的检测流程如图3所示。
1、所述MNP标记法检测肺炎衣原体的检测灵敏度、稳定性分析
如表2所示，所述试剂盒能在10拷贝/反应的样本中稳定的检出7个以上MNP位点，而在0拷贝/反应的少数样本中最多检出1个MNP位点，所述试剂盒能够明显区分10拷贝/反应和0拷贝/反应的样品，具有技术稳定性和低至10拷贝/反应的检测灵敏度。
表2肺炎衣原体的MNP标记法的检测灵敏度、稳定性分析
Figure PCTCN2021129163-appb-000002
2、MNP标记检测试剂盒检测肺炎衣原体的重现性和准确性评估
基于两次重复中，共同检出标记的基因型是否可重现，评估MNP标记检测方法检测肺炎衣原体的重现性和准确性。具体地，对100拷贝样品的12组数据分别进行两两比较，结果如表3所示，主基因型存在差异的MNP标记数目都为0；依据2次重复实验间可重现的 基因型认为是准确的原则，准确率a＝1-(1-r)/2＝0.5+0.5r，r代表重现率，即主基因型可重现的标记数目占共有标记数目的比率。本项目重现性试验中每个样品不同文库间、不同建库批次间MNP标记主基因型的差异对数为0，重现率r＝100％，准确率a＝100％。基于此，所述试剂盒能够准确、灵敏地检测低于10拷贝/反应的肺炎衣原体。
表3肺炎衣原体MNP标记检出方法的重现性和准确率评估
Figure PCTCN2021129163-appb-000003
3、MNP标记检测试剂盒检出肺炎衣原体的阈值判定
如表3所示，在1个拷贝/反应的样本中能检出比对到肺炎衣原体的序列，至少覆盖1个MNP标记。而在部分空白对照中也检出了肺炎衣原体的序列。由于MNP标记检测方法的极度灵敏，因此检测过中的数据污染容易导致假阳性的产生。因此本实例中制定如下质控方案。
质控方案具体如下：
1)测序数据量大于5百万碱基。测算依据是每个样品检测MNP标记的数目是15个，一条测序片段的长度是300个碱基，所以当数据量大于5百万碱基时，大部分样品一次实验可以保证覆盖每个标记的测序片段数量达到1000倍，保证对每个MNP标记碱基序列的精准分析。
2)根据测试样品中的肺炎衣原体的信号指数S和空白对照中肺炎衣原体的噪音指数P判定污染是否可接受，其中：
空白对照噪音指数P＝nc/Nc，其中nc和Nc分别代表空白对照中，肺炎衣原体的测序片段的数量和总测序片段数量。
测试样品的信号指数S＝nt/Nt，其中nt和Nt分别代表测试样品中，肺炎衣原体的测序片段的数量和总测序片段数量。
3)计算测试样品中MNP标记的检出率，指的是检出标记数和总设计标记数的比值。
如表4所示，肺炎衣原体在空白对照中的噪音指数平均值是0.03％，而在1个拷贝的样品中的信号指数平均值是0.06％，1个拷贝的样品和空白对照的信噪比的平均值是2.1，因此，本发明规定当信噪比大于10倍时，可判定检测体系中的污染是可接受的。
如表4所示，在10个拷贝的样品和空白对照的信噪比的平均值是84，在10拷贝/反应的12组数据中，能稳定的检出至少8个MNP标记，占总标记的53.3％。因此，在保证准确性的情况下，本标准规定肺炎衣原体的信噪比判定阈值是40，即当样品中肺炎衣原体的信噪比大于40，且标记检出率大于等于30％时，判定样本中检出了肺炎衣原体的核酸。因此本发明所提供的试剂盒能灵敏的检测到10copy/反应的肺炎衣原体。
表4待测样品中肺炎衣原体的信噪比
Figure PCTCN2021129163-appb-000004
4、MNP标记检测方法检测肺炎衣原体的特异性评估
人为的将肺炎衣原体和结核分枝杆菌、不动杆菌属菌株、霍氏鲍特菌、百日咳鲍特菌、肺炎支原体、EB病毒、流感嗜血杆菌、水痘带状疱疹病毒、巨细胞病毒、单纯疱疹病毒、人博卡病毒、肺炎克雷伯杆菌、军团菌属菌株、卡他莫拉菌、铜绿假单胞菌、立克次氏体属、金黄色葡萄球菌、肺炎链球菌、酿脓链球菌的DNA按照等摩尔量的混在一起，制备混合模板，以空白模板作为对照，采用本发明所提供的方法对混合模板中的肺炎衣原体进行检测，进行3个重复实验。结果在3个重复中获得测序序列都仅能比对到肺炎衣原体的MNP位点，检出的位点数目依次为14，14和15个。按照所述的质控方案和判定阈值进行分析后，在3个重复实验中都特异的检出肺炎衣原体的核酸，表明所述MNP标记和所述试剂盒在复杂模板中检测肺炎衣原体的高特异性。
实施例3、肺炎衣原体菌株间的遗传变异检测
利用所述的试剂盒和MNP标记组合检测方法对湖北省疾控预防控制中心提供的5份肺炎衣原体菌株进行检测，样本依次命名为S1-S5，每个样品的测序平均覆盖倍数达1305倍，每个菌株平均可以检出全部15个MNP标记(表5)。将5个菌株的指纹图谱进行两两比对，结果如表4错误！未找到引用源。所示，有1份(S-1)和同批次一起检测的4份肺炎衣原体均存在部分标记的主基因型差异(表5)，存在菌株间变异，可能属于不同的分离株。
所述的试剂盒通过检测MNP标记鉴定菌株间遗传变异的应用可以用于保证不同实验室相同命名肺炎衣原体菌株的遗传一致性，从而保证研究结果的可比较性，这对于肺炎衣原体的科学研究具有重要意义。而在临床上，可针对差异标记是否影响抗药性斟酌诊断方案。
表5 6个肺炎衣原体的检测分析
Figure PCTCN2021129163-appb-000005
实施例4、肺炎衣原体菌株内部的遗传变异检测
作为群体生物，肺炎衣原体群体内部部分个体发生变异，使群体不再纯合，形成异质的杂合群体，影响尤其是试验用微生物表型的稳定性和一致性。这种变异体在对群体进行分子标记检测时，表现为标记的主基因型外的等位基因型。当变异个体还未累积时，只占群体的极少部分，表现为低频率的等位基因型。低频率的等位基因型往往和技术错误混在一起，导致现有技术难以区分。本发明检测的是高多态性的MNP标记。基于多个错误同时发生的几率低于一个错误发生的几率，MNP标记的技术错误率显著低于SNP标记。
本实施例次等位基因型的真实性评估按如下进行：首先按照以下规则排除具有链偏好性(在DNA双链上覆盖的测序序列数的比值)的等位基因型：链偏好性大于10倍，或者与主等位基因型的链偏好性之差大于5倍。
不存在链偏好性的基因型基于表5测序序列数目和比例判定其真实性。表6列出了基于BINOM.INV函数计算在α＝99.9999％的概率保障下，e max(n＝1)和e max(n≥2)分别为1.03％和0.0994％时，在各个标记中次等位基因型测序序列数目的临界值，只有次等位基因型的测序序列数目超过临界值时判定为真实的次等位基因型。当存在多个候选次等位基因时，对各 候选等位基因型的P值进行多重校正，FDR<0.5％的候选等位基因判定是真实的次等位基因型。
表6涉及到的参数e max(n＝1)和e max(n≥2)指的是携带n个SNP的错误等位基因的测序序列数占该标记总测序序列数的最高比例。e max(n＝1)和e max(n≥2)分别为1.03％和0.0994％是根据在930个纯合MNP标记检测到的所有次等位基因型的频率获得。
表6部分测序深度下进行判定次等位基因型的临界值
Figure PCTCN2021129163-appb-000006
按照上述参数，将S-1的核苷酸按照以下8个比例1/1000，3/1000，5/1000，7/1000，1/100，3/100，5/100，7/100混入S-2的核苷酸中，制备人工杂合样本，每个样本检测3次重复，获得共计24个测序数据。通过和S-1，S-2的MNP标记的基因型进行精准比对，在24个人工杂合样本中均检测到了S-1的基因型，人工杂合样本物中S-1的浓度占比低至1/1000，说明了所开发的肺炎衣原体的MNP标记检测方法在检测菌株群体内部遗传变异的适用性。
实施例5肺炎衣原体DNA指纹数据库的构建
利用常规CTAB法、商业化试剂盒等方法提取用于构建肺炎衣原体DNA指纹数据库的所有菌株或是样本的DNA，采用琼脂糖凝胶和紫外分光光度计检测DNA的质量。若所提取的DNA在260nm与230nm处的吸光度值的比值大于2.0，260nm与280nm吸光度值比值介于1.6与1.8之间，DNA电泳主带明显，无明显降解和RNA残留，则说明基因组DNA达到相关的质量要求，可进行后续实验。
将上述5个菌株的测序数据进行序列比对后获得每个菌株每个标记的主基因型，形成每个菌株的MNP指纹图谱。将每个菌株的MNP指纹图谱录入数据库文件，构成肺炎衣原体的MNP指纹数据库；将在新的样本中检测到的肺炎衣原体的MNP指纹图谱，同构建的MNP指纹数据库进行比对，将主基因型存在差异的样本的MNP指纹图谱录入已构建的MNP指纹数据库。所构建的MNP指纹数据库基于检测的菌株的基因序列，因此和所有的高通量测序数据兼容，具有完全可共建共享、随时可更新的特征。
实施例6、在肺炎衣原体精细分型中的应用
首先构建肺炎衣原体的参考序列库，由已经公开的肺炎衣原体的基因组序列和已构建的肺炎衣原体的DNA指纹数据库组成；利用实施例2所述的引物组合和MNP标记位点检测方法，获得每个待测样品中肺炎衣原体的MNP指纹图谱；将每个菌株的DNA指纹图谱同构建的参考序列库进行比对，筛选获得和序列库中遗传距离最接近的菌株；和已有菌株的基因型100％相同的，为已有的变型，在至少一个MNP位点存在主基因型差异的，为新的变型，实现对肺炎衣原体的精细分型。所检测的5份肺炎衣原体样本中，有1份和其他4份在3个MNP标记的主基因型存在差异，4个指纹图谱一致的菌株和参考序列库中的AR39菌株最相似，相似性高达93.3％。因此，5个菌株在参考序列库中没有基因型完全一致的菌株，属于2个不同的新的变型。可见，所述的方法对肺炎衣原体的分辨率达到了单碱基的水平，可以实现对样本中肺炎衣原体的精细分型。
最后，还需要说明的是，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的 要素。
尽管已描述了本发明实施例的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明实施例范围的所有变更和修改。
显然，本领域的技术人员可以对本发明实施例进行各种改动和变型而不脱离本发明实施例的精神和范围。这样，倘若本发明实施例的这些修改和变型属于本发明实施例权利要求及其等同技术的范围之内，则本发明实施例也意图包含这些改动和变型在内。
Figure PCTCN2021129163-appb-000007
Figure PCTCN2021129163-appb-000008
Figure PCTCN2021129163-appb-000009
Figure PCTCN2021129163-appb-000010
Figure PCTCN2021129163-appb-000011
Figure PCTCN2021129163-appb-000012
Figure PCTCN2021129163-appb-000013
Figure PCTCN2021129163-appb-000014
Figure PCTCN2021129163-appb-000015
Figure PCTCN2021129163-appb-000016
Figure PCTCN2021129163-appb-000017
Figure PCTCN2021129163-appb-000018
Figure PCTCN2021129163-appb-000019

Claims (9)

  1. 一种肺炎衣原体的核心MNP标记组合，其特征在于，所述MNP标记组合包括15个标记，具体的核苷酸序列如SEQ ID NO.1-SEQ ID NO.15所示。
  2. 一种用于检测权利要求1所述肺炎衣原体核心MNP标记组合的多重PCR引物对组合，其特征在于，所述多重PCR引物对组合包括15对引物，具体的引物核苷酸序列如SEQ ID NO.16-SEQ ID NO.45所示。
  3. 一种用于检测权利要求1所述肺炎衣原体MNP标记组合的检测试剂盒，其特征在于，所述试剂盒包括权利要求2所述的引物对组合。
  4. 根据权利要求3所述的检测试剂盒，其特征在于，所述试剂盒还包括多重PCR预混液。
  5. 权利要求1所述的肺炎衣原体的MNP标记组合或权利要求2所述的引物对组合或权利要求3-4任一所述的检测试剂盒在非诊断目的的肺炎衣原体定性检测中的应用。
  6. 权利要求1所述的肺炎衣原体的MNP标记组合或权利要求2所述的引物对组合或权利要求3-4任一所述的检测试剂盒在制备肺炎衣原体定性检测产品中的应用。
  7. 权利要求1所述的肺炎衣原体的MNP标记组合或权利要求2所述的引物对组合或权利要求3-4任一所述的检测试剂盒在 检测肺炎衣原体菌株内部和菌株间遗传变异中的应用。
  8. 权利要求1所述的肺炎衣原体的MNP标记组合或权利要求2所述的引物对组合或权利要求3-4任一所述的检测试剂盒在构建肺炎衣原体数据库中的应用。
  9. 权利要求1所述的肺炎衣原体的MNP标记组合或权利要求2所述的引物对组合或权利要求3-4任一所述的检测试剂盒在肺炎衣原体精细分型检测中的应用。
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