WO2022247284A1 - Maldi-tof ms检测生殖道病原体的检测产品及用途 - Google Patents

Maldi-tof ms检测生殖道病原体的检测产品及用途 Download PDF

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WO2022247284A1
WO2022247284A1 PCT/CN2021/143454 CN2021143454W WO2022247284A1 WO 2022247284 A1 WO2022247284 A1 WO 2022247284A1 CN 2021143454 W CN2021143454 W CN 2021143454W WO 2022247284 A1 WO2022247284 A1 WO 2022247284A1
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seq
fragment
extension
pcr amplification
detection
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李维
邬文燕
张雪莲
张海燕
马庆伟
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北京毅新博创生物科技有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Definitions

  • the invention belongs to the field of molecular biology detection, and relates to a detection product and application for quickly identifying 10 kinds of reproductive tract pathogens by using laser time-of-flight mass spectrometry technology.
  • the most common disease is not a cold, but a reproductive tract infection, and the incidence rate is as high as 90%; the most common symptoms are not fever, cough, headache, etc. Vaginal itching, odor, pain, frequent urination, urgency, etc.
  • the occurrence of many other diseases in women is also closely related to the abnormal function of the reproductive system, such as osteoporosis, coronary heart disease, diabetes, and chloasma.
  • the problem of the reproductive system is related to the health and happiness of a woman's life, and it can also be said to be the "source of all diseases" for women.
  • Female genital tract infection is an infectious disease caused by pathogenic microorganisms such as bacteria, viruses, and parasites that occurs in women from the vulva to the uterus, mainly including vaginitis (candida, trichomonas, senile, bacterial Sexual vaginosis), gonorrhea, cervicitis (polyp, Nasser's cyst), pelvic inflammatory disease, etc. are common diseases in women, and almost every adult woman and some girls have had it.
  • Female reproductive tract infection has the characteristics of high prevalence, high proportion of asymptomatic, high proportion of non-seeking doctors and high proportion of unreasonable treatment, leading to various serious complications and sequelae. The level of female reproductive health directly affects the quality of the population and cannot be ignored.
  • Female reproductive tract infection has become one of the most serious infectious diseases in the world. Factors related to female reproductive infection Due to the influence of various factors such as anatomy, physiology, sexual activity, childbirth and hygiene habits, the female reproductive tract is prone to various infections
  • Reproductive tract infection is a common disease in gynecology, caused by the invasion of bacteria, viruses, Candida albicans, protozoa, mycoplasma, chlamydia and other pathogens, including vulvitis, vaginitis, cervicitis and upper reproductive tract infection of female lower reproductive tract. pelvic inflammatory disease of the tract. Reproductive tract infection has the characteristics of wide prevalence, high recurrence rate, and low consultation rate. It is the most common gynecological disease, and the domestic prevalence rate is as high as 42.9%.
  • genital tract infection is not timely diagnosed and treated correctly, it can increase the risk of sexually transmitted disease infection, which can cause abnormal uterine bleeding, infertility, ectopic pregnancy, chronic pelvic pain, miscarriage, premature rupture of membranes, premature delivery, and stillbirth , Stillbirth and congenital infection and other complications and sequelae, not only endanger the health of women, but also endanger the fetus and newborn. Reproductive tract infection has become a social public health problem that seriously affects women's health and brings serious burdens to families and society.
  • HPV high-risk human papillomavirus
  • Reproductive tract pathogens are one of the important micro-ecological systems of the human body. They form a mutually restrictive and coordinated micro-ecological balance with the host and the environment. This dynamic balance plays a key role in maintaining the health of the female reproductive tract.
  • Female genital tract pathogens include Staphylococcus aureus, Hemolytic Streptococcus, Neisseria gonorrhoeae, Group B Streptococcus, Escherichia coli, Gardnerella vaginalis, Candida albicans, Mycoplasma, Chlamydia trachomatis and HPV.
  • the commonly used pathogen detection methods include real-time fluorescent quantitative PCR method, PCR-capillary electrophoresis method, and high-throughput sequencing method.
  • the detection throughput of real-time fluorescent quantitative PCR method is low, and it is difficult to detect multiple pathogenic bacteria at the same time.
  • PCR-capillary electrophoresis has greatly improved in terms of automation and analysis time, its detection throughput is far lower than that of emerging molecular biology detection methods in recent years.
  • High-throughput sequencing (NGS) can sequence hundreds of thousands to millions of DNA molecules at a time, and plays an important role in the detection of clinical pathogens.
  • NGS technology requires high equipment requirements, high technical thresholds, and a large amount of funds for platform construction Investment, it is difficult to promote in the field of clinical application. Therefore, in the field of clinical application, it is urgent to establish a rapid, accurate, sensitive, and high-throughput detection system for reproductive tract pathogens, so as to provide sufficient basis for the diagnosis, treatment, and prognosis of diseases.
  • Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, referred to as MALDI-TOF MS) technology is a mass spectrometry analysis technology that came out in the late 1980s and developed rapidly. Its mass analyzer is an ion drift tube (ion dirt tube). The ions generated by the ion source are collected first, and all ion velocities in the collector become 0.
  • ions of different masses can be separated according to the mass-to-charge ratio, and the molecular mass and purity of biological macromolecules such as peptides, proteins, nucleic acids, and polysaccharides can be accurately detected. It has high accuracy, strong flexibility, and large throughput. , short detection cycle, and high cost performance.
  • the early MALDI-TOF MS mass spectrometry technology was used to detect clinical microorganisms, mainly for the characteristic proteins of microorganisms.
  • Lin Haoyun Application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry in clinical microbial identification", “Chinese Journal of Zoonotic Diseases", No. 8, 2014
  • protein composition is rarely affected by environmental conditions, mainly determined by genetic characteristics, so MALDI-TOF MS is more accurate and reliable.
  • MALDI-TOF MS A major advantage is the universality of its identification of microorganisms. It can analyze a large number of targets simultaneously and does not require the selection of specific recognition antibodies or primers. For microorganisms with genetic mutations, MALDI-TOF MS can at least identify them to the nearest neighbor database middle". However, the report states that while components of 2–3 species of bacteria can be detected, “the identification capability of MALDI-TOF MS is significantly limited when a single species is strongly dominant in the mixture”.
  • This method uses conventional processing (processing by absolute ethanol, formic acid and acetonitrile, and supplemented by centrifugation, and finally draws the supernatant for detection), although it can characterize the characteristic map of the bacteria to a certain extent, it is still pending.
  • the test object contains proteins, lipids, lipopolysaccharides and lipooligosaccharides, DNA, polypeptides and other molecules that can be ionized.
  • the amount of information in the map is too large, and the characteristics of the map are low due to the large number of molecules to be detected. It is only applicable to a specific bacterium and cannot be extended to other large numbers of bacterial detection.
  • this diagnostic model can be used to detect Escherichia coli, Staphylococcus aureus and Candida albicans, since this method needs to find out the characteristic curve peaks from a large number of characteristic polypeptide fragments, and the polypeptide fragments of each bacteria interfere with each other, Therefore, there is a certain influence on the detection effect.
  • Chinese patent application 201210272533.6 "Method for Establishing Helicobacter Pylori Nucleic Acid Fingerprint and Its Products” discloses a method for rapid identification of Helicobacter pylori based on mass spectrometry technology, including PCR amplification, SAP enzyme digestion, transcription and nuclease digestion, and purification , mass spectrometer detection and other steps.
  • the method utilizes time-of-flight mass spectrometry technology to detect nucleic acid fragments with different molecular weights and abundances and form a spectrum.
  • nucleic acid fragments after the nucleic acid fragments are amplified by PCR, they need to undergo SAP enzyme digestion, transcription and nuclease digestion, which can only identify the change of a single base, and cannot detect long fragments of DNA with characteristic sequences.
  • nucleic acid detection methods have been developed, such as the hME and iPLEX methods of Agena Company in the United States, the GOOD assay method of Bruker Company in Germany, and the RFMP method of GeneMatrix Company in South Korea.
  • various companies tend to detect oligonucleotide fragments with smaller molecular weights when detecting target sites.
  • the RFMP method uses multiplex PCR for sites containing single nucleotide polymorphisms (SNPs).
  • the product is subjected to restriction enzyme digestion to produce oligonucleotide fragments of about 2000-4000 Da for detection, while the GOOD assay method uses phosphodiesterase (Phosphodiesterase, PDE) to cut the oligonucleotide fragments containing SNP sites into 1000-Da Small fragments around 2000 Da are detected.
  • PDE phosphodiesterase
  • one of the principles of the present invention is that the present invention provides a combination of PCR technology, single base extension technology and mass spectrometry detection technology to detect the characteristic map of the characteristic fragments related to the typing of the pathogen to be tested .
  • 10 primer sets were designed for 10 pathogens (Staphylococcus aureus, Group B Streptococcus, Neisseria gonorrhoeae, Gardnerella vaginalis, Candida albicans, Mycoplasma genitalium, Ureaplasma urealyticum, Chlamydia trachomatis and HPV16 and HPV18) , in multiplex PCR, simultaneously amplify the DNA fragment products containing specific target sequences of 10 pathogens, and perform single-base extension on the above-mentioned products through high-efficiency single-base primers, that is, the extension primers are respectively extended at 10 specific sites A nucleotide such that the type of nucleotide extended, respectively, correlates with the genotype at that site.
  • pathogens Staphylococcus aureus, Group B Streptococcus, Neisseria gonorrhoeae, Gardnerella vaginalis, Candida albicans, Mycoplasma genitalium
  • the extension product is purified, and the purified mixture to be detected is detected by MALDI TOF MS mass spectrometry, and the molecular weight of each substance in the mixture to be detected is determined through the mass spectrum peak, and compared with the theoretical molecular weight of the pre-calculated amplification product, thereby Determine the type of pathogen to be detected in the extension product.
  • the second principle of the present invention is that, in order to solve the mutual interference of multiple PCR products and exclude unsuitable sites to be detected, the present invention conducts optimized screening to determine the optimized combination of amplification products and extension primers.
  • the third principle of the present invention is that in the further optimization process of the multiplex PCR system, in order to make the molecular weights of the extension primers and extension products distributed in the ideal mass spectrometry detection window (that is, 4000-9000Da), the introduction of the amplification primers in the present invention does not affect
  • the tag sequence amplified by PCR makes the final 10 kinds of extension products fall into the detection window, and at the same time distinguish each other, avoiding the overlapping of molecular weights, thereby improving the detection accuracy and sensitivity.
  • the first object of the present invention is to provide a kind of MALDI TOF-MS to detect 10 kinds of genital tract pathogen characteristic fragment primer composition, it comprises:
  • the aforementioned PCR amplification primer sequence is a core sequence, which may include a protective base sequence at the 5' end, preferably 5-15 bases.
  • the protected base sequence is selected from adding a 10bp tag (ACGTTGGATG) to the 5' section, for example, the PCR primer SEQ ID NO: 1 is 5'-ACGTTGGATGGCCGTTATCTGTTTGTGATG-3'.
  • the 5' end of the extension primer may also increase a base sequence as a linker.
  • the second object of the present invention is to provide a detection product prepared by the above primer composition, which can detect 10 kinds of characteristic fragments related to the detection of reproductive tract pathogens by MALDI TOF MS, wherein the characteristic fragments of the pathogen and Corresponding PCR amplification primers and extension primers are selected from:
  • the nuc fragment of Staphylococcus aureus and the corresponding PCR amplification primers are SEQ 1-2, and the extension primer is SEQ 21;
  • the scpB fragment of Group B Streptococcus and the corresponding PCR amplification primers are SEQ 3-4, and the extension primers are SEQ 22;
  • the NG fragment of Neisseria gonorrhoeae and the corresponding PCR amplification primers are SEQ 5-6, and the extension primer is SEQ 23;
  • the 16sRNA fragment of Gardnerella vaginalis and the corresponding PCR amplification primers are SEQ 7-8, and the extension primer is SEQ 24;
  • the ITS fragment of Candida albicans and the corresponding PCR amplification primers are SEQ 9-10, and the extension primer is SEQ 25;
  • the PA fragment of Mycoplasma genitalium and the corresponding PCR amplification primers are SEQ 11-12, and the extension primer SEQ 26;
  • the UreA fragment of Ureaplasma urealyticum and the corresponding PCR amplification primers are SEQ 13-14, and the extension primer is SEQ 27;
  • the PmpF fragment of Chlamydia trachomatis and the corresponding PCR amplification primers are SEQ 15-16, and the extension primer is SEQ 28;
  • the L1 fragment of HPV16 and the corresponding PCR amplification primers are SEQ 17-18, and the extension primer is SEQ 29;
  • the L1 fragment of HPV18 and the corresponding PCR amplification primers are SEQ 19-20, and the extension primer is SEQ 30.
  • the product is a test kit comprising:
  • Reaction solution I for PCR amplification including: PCR amplification primers, high temperature resistant DNA polymerase, dNTPs, PCR reaction buffer (comprising dNTPs, Tris-HCl, MgCl 2 );
  • Reaction solution II for PCR product purification which contains Tris-HCl, MgCl 2 ;
  • Reaction solution III for single base extension reaction including: extension primers, high temperature resistant single base elongation enzyme, ddNTPs, extension reaction buffer containing Tris-HCl and MgCl 2 .
  • the kit can also include: negative quality control, positive quality control, resin for purification, target chip for spotting and mass spectrometry detection, exonuclease, human genomic DNA extraction reagent and other reagents.
  • reaction system of the PCR amplification contains:
  • the concentration of each primer pair is controlled between 0.5-2 ⁇ M.
  • the PCR amplification reaction program was as follows: pretreatment at 50°C for 2 minutes, then pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, and 45 cycles; extension at 72°C for 5 min.
  • the SAP enzyme digestion reaction process add 1.5 ⁇ L of reaction solution II and 0.5 ⁇ L of enzyme II to 5 ul of the amplified product, and cycle at 37° C. for 30 minutes and at 65° C. for 5 minutes.
  • Extension reaction process add 0.83 ⁇ L reaction solution II, 0.23 ⁇ L enzyme II, 0.94 ⁇ L extension primer to the digestion product, and perform one cycle at 94°C for 30s; 5s at 94°C, followed by 5 cycles of 5s and 80°C at 56°C 5s at °C, a total of 40 cycles; finally at 72°C for 5 minutes, a total of 1 cycle.
  • the third object of the present invention is to use the above primer composition and detection product to detect the characteristic fragment method of 10 kinds of reproductive tract pathogens, including the following steps:
  • Multiplex PCR use specific PCR amplification primers to simultaneously amplify the target DNA regions of 10 reproductive tract pathogens in one reaction system to obtain PCR products containing the DNA regions of 10 specific sites;
  • step (1) PCR product purification: the PCR product obtained in step (1) is purified to reduce interference to subsequent reactions;
  • Mass spectrometer detection point the purified product obtained in step (4) on the target sheet containing the matrix, and put it into a mass spectrometer for detection;
  • the characteristic fragment of described pathogen and corresponding PCR amplification primer and extension primer are selected from:
  • the nuc fragment of Staphylococcus aureus and the corresponding PCR amplification primers are SEQ 1-2, and the extension primer is SEQ 21;
  • the scpB fragment of Group B Streptococcus and the corresponding PCR amplification primers are SEQ 3-4, and the extension primers are SEQ 22;
  • the NG fragment of Neisseria gonorrhoeae and the corresponding PCR amplification primers are SEQ 5-6, and the extension primer is SEQ 23;
  • the 16sRNA fragment of Gardnerella vaginalis and the corresponding PCR amplification primers are SEQ 7-8, and the extension primer is SEQ 24;
  • the ITS fragment of Candida albicans and the corresponding PCR amplification primers are SEQ 9-10, and the extension primer is SEQ 25;
  • the PA fragment of Mycoplasma genitalium and the corresponding PCR amplification primers are SEQ 11-12, and the extension primer SEQ 26;
  • the UreA fragment of Ureaplasma urealyticum and the corresponding PCR amplification primers are SEQ 13-14, and the extension primer is SEQ 27;
  • the PmpF fragment of Chlamydia trachomatis and the corresponding PCR amplification primers are SEQ 15-16, and the extension primer is SEQ 28;
  • the L1 fragment of HPV16 and the corresponding PCR amplification primers are SEQ 17-18, and the extension primer is SEQ 29;
  • the L1 fragment of HPV18 and the corresponding PCR amplification primers are SEQ 19-20, and the extension primer is SEQ 30.
  • the purification process in step 2 can be selected from alkaline phosphatase digestion, alkaline phosphatase and exonuclease ExoI digestion, gel cutting purification, PCR purification column passing and the like.
  • high temperature enzyme inactivation treatment is performed after purification using alkaline phosphatase digestion, or alkaline phosphatase and exonuclease ExoI digestion.
  • the aforementioned PCR primer sequence is a core sequence, which may include a protective base sequence at the 5' end, preferably 5-15 bases.
  • the protected base sequence is selected from adding a 10bp tag (ACGTTGGATG) to the 5' section, for example, the PCR primer SEQ ID NO: 1 is 5'-ACGTTGGATGGCCGTTATCTGTTTGTGATG-3'.
  • extending the 5' end of the primer can also increase the base sequence as a linker.
  • the method can be used for non-diagnostic purposes, and is widely used in the identification of pathogens in the fields of environmental sanitation, food safety testing, import and export inspection and quarantine, so as to maintain public health safety.
  • the fourth object of the present invention is to provide a method for preparing a nucleic acid fingerprint library for identifying reproductive tract pathogens by mass spectrometry, comprising the following steps:
  • the specific primer set of the nuc target fragment of Staphylococcus aureus is selected from: SEQ1 and SEQ2;
  • the specific primer set of the target fragment of the scpB fragment of Group B Streptococcus is selected from: SEQ3 and SEQ4;
  • the specific primer set of the target fragment of the NG fragment of Neisseria gonorrhoeae is selected from: SEQ5 and SEQ6;
  • the specific primer set of the target fragment of the 16sRNA fragment of Gardnerella vaginalis is selected from: SEQ7 and SEQ8;
  • the specific primer set of the target fragment of the ITS fragment of Candida albicans is selected from: SEQ9 and SEQ10;
  • the specific primer set of the PA fragment target fragment of Mycoplasma genitalium is selected from: SEQ11 and SEQ12;
  • the specific primer set of the UreA fragment target fragment of Ureaplasma urealyticum is selected from: SEQ13 and SEQ14;
  • the specific primer set of the target fragment of the PmpF fragment of Chlamydia trachomatis is selected from: SEQ15 and SEQ16;
  • the specific primer set of the target fragment of the L1 fragment of HPV16 is selected from: SEQ17 and SEQ18;
  • the specific primer set of the target fragment of the L1 fragment of HPV18 is selected from: SEQ19 and SEQ20;
  • the extension primer of the nuc target fragment of Staphylococcus aureus is selected from: SEQ21;
  • the extension primer of the scpB fragment target fragment of Group B Streptococcus is selected from: SEQ22;
  • the extension primer of the target fragment of the NG fragment of Neisseria gonorrhoeae is selected from: SEQ23;
  • the extension primer of the target fragment of the 16sRNA fragment of Gardnerella vaginalis is selected from: SEQ24;
  • the extension primer of the ITS fragment target fragment of Candida albicans is selected from: SEQ25;
  • the extension primer of the PA fragment target fragment of Mycoplasma genitalium is selected from: SEQ26;
  • the extended primer of the UreA fragment target fragment of Ureaplasma urealyticum is selected from: SEQ27;
  • the extension primer of the target fragment of the PmpF fragment of Chlamydia trachomatis is selected from: SEQ28;
  • the extended primer of the L1 fragment target fragment of HPV16 is selected from: SEQ29;
  • the extension primer of the L1 fragment target fragment of HPV18 is selected from: SEQ30;
  • step (7) Compare and analyze the nucleic acid fingerprint characteristic spectrum obtained in step (6) with the blank control, negative control, and positive control by computer software, and obtain the standard nucleic acid fingerprint characteristic spectrum of 10 kinds of reproductive tract pathogens.
  • the molecular weights of the specific fragments and the characteristic peaks of the mass spectrum corresponding to the nucleic acid fingerprint characteristic spectra of the above-mentioned 10 kinds of target fragments are respectively:
  • the target fragment of the scpB fragment of Group B Streptococcus 5447.6 (m/z);
  • the target fragment of the NG fragment of Neisseria gonorrhoeae 5288.3 (m/z);
  • Target fragment of 16sRNA fragment of Gardnerella vaginalis 5919.8 (m/z);
  • the target fragment of the ITS fragment of Candida albicans 6363.2 (m/z);
  • the target fragment of the PA fragment of Mycoplasma genitalium 6694.4 (m/z);
  • the target fragment of the UreA fragment of Ureaplasma urealyticum 5997.9 (m/z);
  • the target fragment of the PmpF fragment of Chlamydia trachomatis 6986.6 (m/z);
  • Target fragment of L1 fragment of HPV16 8241.3 (m/z);
  • Target fragment of L1 fragment of HPV18 5424.6 (m/z).
  • the solid phase support of the DNA adsorption column includes but is not limited to gel, resin, silica, silica gel, magnetic beads, glass powder, glass beads and the like.
  • the matrix is a composite matrix comprising an acidic component including, but not limited to, formic acid, acetic acid, and citric acid.
  • the chip is a dedicated microarray chip for time-of-flight mass spectrometry, and its material includes but not limited to stainless steel, diamond, single crystal silicon, and quartz crystal.
  • step (2) the NanoDrop ND-2000 nucleic acid detector is used to measure the concentration of plasmid DNA, and the copy number of DNA is determined as a standard for sensitivity to quantify the mother liquor.
  • the aforementioned PCR amplification primer sequence is a core sequence, which may include a protective base sequence at the 5' end, preferably 5-15 bases.
  • the protected base sequence is selected from adding a 10bp tag (ACGTTGGATG) to the 5' section, for example, the PCR primer SEQ ID NO: 1 is 5'-ACGTTGGATGGCCGTTATCTGTTTGTGATG-3'.
  • the 5' end of the extension primer may also increase a base sequence as a linker. .
  • the mass spectrometer is a MALDI TOF MS mass spectrometer.
  • the software is the BioExplore software researched and developed by the inventor himself, whose copyright number is Ruan Zhu Deng Zi No. 136879 and registration number 2009SR10700.
  • the present invention has the following advantages:
  • the present invention proposes for the first time to use multiplex PCR combined with clinical mass spectrometry to realize multiple detection of specific fragments related to 10 reproductive tract pathogens, which has extremely high biological value.
  • Sensitive the present invention integrates technologies such as multiplex PCR, single base extension, and mass spectrometry detection. It can not only amplify the detection template through PCR technology, but also detect trace samples through mass spectrometry technology. Much better than using PCR alone to detect pathogen-specific fragments, so its detection sensitivity is high.
  • Single-base extension also known as “micro-sequencing”
  • special, different from Sequencing technology extends hundreds of bases, this technology only extends a single base, and the probability of error is lower;
  • the present invention completes the detection of related pathogens by identifying the characteristic maps of specific sites related to pathogen typing.
  • the invention overcomes the defect of too few types of pathogens detected at one time in the prior art, and has low cost.
  • the data analysis required by the present invention is simple, only need to observe the spectrogram, and no complex bioinformatics analysis is required.
  • the invention is low in cost, does not require fluorescent labels, and reduces system complexity and signal interpretation errors caused by the addition of fluorescent chemical probes.
  • the present invention provides a combined multiple PCR, single base extension and mass spectrometry detection technology to detect the characteristic maps of specific fragments or sites of 10 reproductive tract pathogens, and then determine the detection scheme of the pathogen type to be detected.
  • the product of the multiplex PCR in the previous step is sequentially purified and multiplexed by single-base extension.
  • there are 10 extension primers which correspond to 10 specific sites respectively, and extend a nucleotide at the corresponding specific site, and the nucleotide is complementary to the genotype at the specific site (such as a specific site).
  • Dots are A genotypes, T nucleotides will be extended on the corresponding extension primers).
  • ddNTPs are used instead of dNTPs, so the extension primer will stop the extension after extending one base.
  • the single base extension product is purified, spotted to the target chip containing the matrix, and excited by the laser in the vacuum environment, and then passed through the flight tube to the detector.
  • the time for different substances to pass through the flight tube is negatively correlated with their molecular weight, that is, the larger the molecular weight, the slower the flight speed and the later the time to reach the detector.
  • the term "protective base” refers to an additional base added at the 5' end of a PCR primer. Since the sequence of the protective base increases the molecular weight of the PCR primer (ie, the core primer), it can prevent the remaining PCR primer from the reaction from entering the mass spectrometry detection window, so as to avoid interference with the detection effect. In addition, the 5' end of the extension primer can also increase the base sequence in an appropriate amount, but its function is not like the protection base of the PCR primer to make it exceed the detection window, but to properly adjust the molecular weight of the extension primer so that the extension primer and its products within a reasonable position within the detection window.
  • extension primers and products corresponding to two gene polymorphic sites are close, by adding bases to one of the extension primers, the molecular weight of the primer and its products will be changed, and the molecular weight of the other extension primers and products will be pulled. Large gap to avoid interference and unclear resolution caused by excessive concentration of mass spectrum peaks in local areas, thereby improving the detection effect. Therefore, the molecular weights of the extended primers and products after adding bases must not exceed the detection window.
  • the above-mentioned extra bases of the extension primers may be referred to as primer adapters.
  • alkaline phosphatase digestion its function is to degrade the residual dNTP in the system after the PCR reaction, the principle is to convert the 5'-P end of the dNTP into a 5'-OH end, thereby losing the ability to bind with the primer and extend the primer , avoiding the impact on the next step of single base extension.
  • the term "digestion with exonuclease ExoI” is to sequentially catalyze the hydrolysis of the 3,5-phosphodiester bonds between the dNTPs that make up the DNA from one end of the single-stranded DNA, so that the single-stranded DNA is finally hydrolyzed into dNTPs. In this technical scheme, it is used to degrade the remaining PCR primers after the PCR reaction. Since the exonuclease can excise the single-stranded PCR primer and will not appear in the detection window, when using this exonuclease, the PCR primer used does not need to include protective bases.
  • single base extension also known as mini sequence, refers to adding an extension primer and ddNTP to the system, and ddNTP is connected to the 3' end of the extension primer to form an extension product (that is, the primer is extended by one base ), according to the principle of complementary base pairing, the genotype at a specific site determines which ddNTP to connect to. This process is similar to the dNTP in the PCR process, which is added to the PCR primer one by one according to the base composition of the complementary chain. Because “ddNTP” is different from ordinary dNTP in that a ddNTP is connected at the despecific site, and cannot be continuously extended like PCR, so it is called single-base extension.
  • Single-base extension is very similar to the sequencing process.
  • a mixture of dNTPs and ddNTPs is added to the sequencing system.
  • the sequencing primers will continue to extend after connecting dNTPs. Only after connecting ddNTPs can the extension be terminated. Therefore, sequencing produces nuclei of different lengths.
  • detection product refers to any conventional product used to detect the genotype of a specific locus, including: detection reagents, detection chips, detection carriers, and detection kits.
  • ddNTP is a special kind of nucleotide.
  • nucleotide there are four kinds of nucleotides used in this technical solution, and there are molecular weight differences among them.
  • the molecular weights of ddATP, ddCTP, ddGTP, and ddTTP are 271.2Da, 247.2Da, 287.2Da, and 327.1 Da (where ddTTP is the modified molecular weight).
  • the corresponding extension primer is 22 bases in length (molecular weight 7200Da)
  • the extension primer when the site is A genotype, the extension primer will extend a T nucleotide and stop the extension , forming an extension product with a length of 23 bases and a molecular weight of 7487.2 Da.
  • the extension primer When the site is of the G genotype, the extension primer will extend a C nucleotide and terminate the extension, forming an extension product with a length of 23 bases and a molecular weight of 7447.2 Da product, there is a 40 Da molecular weight difference between the two products. That is, for this locus, if the extended primer of 7200Da is used, the G genotype will correspond to the mass spectrum peak of 7447.2Da, and the A genotype will correspond to the mass spectrum peak of 7487.2Da.
  • purification refers to the processing steps used to reduce the influence of other substances in the system to be tested on subsequent reactions.
  • gel cutting purification, purification column, etc. are used to separate impurities by electrophoresis, purification column, etc., and recover relatively pure PCR products. It can be considered as the first purification method. This method is generally time-consuming and complicated to operate.
  • alkaline phosphatase When the sample size is large; the role of alkaline phosphatase is to degrade (also known as "digest") dNTP, so that it cannot continue to participate in PCR or single-base extension reaction as a substrate for DNA polymerase or single-base elongation enzyme, so as not to Interfering with subsequent reactions can be considered as the second purification method. It should be noted that the exonuclease ExoI alone does not play a role in purification.
  • m/z mass-to-charge ratio is the ratio of mass to charge.
  • the ratio of the maximum molecular mass to the minimum charge (that is, one charge) obtained in the spectrum can also be expressed as molecular weight (Da, that is, relative molecular weight) .
  • detection window refers to the range that can be used to detect the molecular weight of nucleotides by mass spectrometry, and usually involves the design reference range of primers.
  • extension primers and extension products with different molecular weights can be designed to avoid different extension primers. There is interference between the product and the product due to the close molecular weight, so that the detection of multiple specific sites can be realized in a relatively wide detection window, such as 4000-9000Da.
  • nucleic acid mass spectrometry for example, it needs to amplify a fragment containing a specific site by PCR reaction first, and then extend the base of the specific site by extending the primer; the PCR of each specific site There is no obvious interference between the reaction and the extension reaction; the molecular weight difference between the extension primers and products of each specific site must be large enough to achieve distinction, etc. Therefore, not all sites can be used for nucleic acid mass spectrometry detection, nor can they Not all primers designed for specific loci can be used for multiplex PCR reactions and multiplex single base extension reactions. For example, Cláudia M.B et al.
  • the scale is mainly limited by the degree of interaction between primers, which affects the detection process of nucleic acid mass spectrometry; in addition, in order to accurately distinguish the differences between different bases, especially adenine (A) and thymine (T) (four bases The difference between the molecular weights of these two bases is the smallest, which is 9Da), and the length of the required oligonucleotides generally does not exceed 40 bases.
  • the molecular weight range of the mass spectrometry detection window is generally 4000-9000Da, that is, The molecular weights of the extension primers and products involved should be distributed within the range of 4000-9000 as much as possible. At the same time, overlap between each extension primer and its extension product should be avoided. It can be seen that not all specific sites can be applied to the detection of nucleic acid mass spectrometry, especially multiple nucleic acid mass spectrometry, and its actual effect will be affected by various experimental factors. Therefore, experiments are needed to verify the feasibility of specific sites and to screen different combination of primers.
  • Figure 1- Figure 13 each group of blank control, negative control, positive control and waiting group for nucleic acid mass spectrometry detection of pathogen DNA to be tested The mass spectrometry result graph of the measured sample, where the arrow shows,
  • Figure 1 is the characteristic peak corresponding to the nuc fragment (6077Da) of Staphylococcus aureus: 6077m/z
  • Figure 2 is the characteristic peak corresponding to the scpB fragment fragment (5447.6Da) of Group B Streptococcus: 5447.6m/z
  • Fig. 3 is the characteristic peak corresponding to the NG fragment object fragment (5288.3Da) of Neisseria gonorrhoeae: 5288.3m/z
  • FIG. 4 is the corresponding characteristic peak of the 16sRNA fragment object fragment (5919.8Da) of Gardnerella vaginalis : 5919.8m/z;
  • Figure 5 is the characteristic peak corresponding to the ITS fragment objective fragment (6363.2Da) of Candida albicans: 6363.2m/z;
  • Figure 6 is the characteristic corresponding to the PA fragment objective fragment (6694.4Da) of Mycoplasma genitalium Peak: 6694.4m/z;
  • Fig. 7 is the corresponding characteristic peak of the UreA fragment object fragment (5997.9Da) of Ureaplasma urealyticum: 5997.9m/z;
  • FIG. 8 is corresponding to the PmpF fragment object fragment (6986.6Da) of Chlamydia trachomatis Characteristic peak: 6986.6m/z;
  • Figure 9 is the characteristic peak corresponding to the L1 fragment target fragment (8241.3Da) of HPV16: 8241.3m/z;
  • Figure 10 is the characteristic peak corresponding to the L1 fragment target fragment (5424.6Da) of HPV18 : 5424.6m/z;
  • Figure 11 is the positive control, from left to right: the characteristic peak corresponding to the Neisseria gonorrhoeae NG fragment (5288.3Da): 5288.3m/z; the characteristic corresponding to the HPV18L1 fragment (5424.6Da) Peak: 5424.6m/z; characteristic peak corresponding to group B Streptococcus scpB fragment (5447.6Da): 5447.6m/z; characteristic peak corresponding to Gardnerella vaginalis 16sRNA fragment (5919.8Da)
  • Figure 14- Figure 30 The mass spectrometry results of the patient's pathogen DNA in Example 4, where the arrows show, Figure 14N1, the detection peaks (m/z) are: 5919.8, 6363.2, 5997.9; Figure 15N2, the detection peaks (m /z) are respectively: 6363.2;
  • Figure 16N3 the detection peaks (m/z) are: 6694.4; Figure 17N4, the detection peaks (m/z) are: 5919.8, 6363.2; Figure 18 N5, the detection peaks (m/z) are: 5919.8; Figure 19N6 , the detection peak value (m/z) is respectively: 8241.3; Fig. 20N7, the detection peak value (m/z) is respectively: 6363.2; Fig. 21N8, the detection peak value (m/z) is respectively: 6363.2; Fig. 22N9, the detection peak value (m/z) is respectively: 6363.2; /z) are respectively: 5288.3; Fig.
  • the detected peak values (m/z) are respectively: 6363.2, 6077; Fig. 24N11, the detected peak values (m/z) are respectively: 6077; Fig. 25N12, the detected peak values (m/z) They are: 5424.6; in Figure 26N13, the detected peak values (m/z) are: 5919.8, 6363.2 respectively; in Figure 27N14, the detected peak values (m/z) are respectively: 6986.6; in Figure 28N15, the detected peak values (m/z) are respectively: 5447.6, 6363.2; Figure 29N16, the detection peak values (m/z) are: 6694.4; Figure 30N17, the detection peak values (m/z) are: 6363.2, 6077;
  • Figure 31- Figure 33 Mass spectrometry results of the blank control, negative control, and positive control in Example 4.
  • the spectrograms are all the peak positions of the extended primers, and there is no positive target characteristic peak (extension product peak), that is, no positive pathogens were detected
  • the spectrograms are all the peak positions of the extended primers , no positive target characteristic peak (extension product peak), that is, no positive pathogen was detected
  • Figure 33 positive control from left to right is: Neisseria gonorrhoeae NG fragment (5288.3Da) corresponding to the characteristic peak: 5288.3m/ z; the characteristic peak corresponding to the HPV18L1 fragment (5424.6Da): 5424.6m/z; the characteristic peak corresponding to the group B Streptococcus scpB fragment (5447.6Da): 5447.6m/z; the Gardnerella vaginalis 16sRNA fragment (5919
  • All detection target regions in the present invention are single-region detection, and the PCR primers correspond to the 3' ends of the above-mentioned reference PCR primers to ensure amplification.
  • the properties were adjusted without affecting the specificity of the primers and without increasing the non-specificity of the primers.
  • the target fragment of Staphylococcus aureus is selected from the nuc fragment, > DQ399678.1:1-450 Staphylococcus aureus nuclease (nuc) gene, partial cds, its sequence number is DQ399678.1;
  • the target segment of group B streptococcus is selected from the scpB segment: >CP053027.1:1577249-1577698 Streptococcus agalactiae strain 01173chromosome, complete genome, and its sequence number is CP053027.1;
  • the target fragment of Neisseria gonorrhoeae is selected from the NG fragment, >AP023076.1:1148-1597Neisseria gonorrhoeae TUM16691plasmid pMTY16691DNA, complete genome, and its sequence number is AP023076.1;
  • the target fragment of Gardnerella vaginalis is selected from the 16sRNA fragment, >L08167.1 Gardnerella vaginalis 3'end of 16S ribosomal RNA, internal transcribed spacer, and 5'end of 23S ribosomal RNA, its sequence No. L08167.1;
  • the target fragment of Candida albicans is selected from the ITS fragment,>MK805514.1:77-526Candida albicans strain TBS611small subunit ribosomal RNA gene,partial sequence;internal transcribed spacer 1,5.8S ribosomal RNA gene,and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNAgene, partial sequence, its sequence number is MK805514.1;
  • the target segment of Mycoplasma genitalium is selected from the PA segment >KP318805.1:413-862Mycoplasma genitalium strain Seattle1MgpB adhesive gene, complete cds, and its sequence number is KP318805.1;
  • the target fragment of Ureaplasma urealyticum is selected from the UreA fragment, >X51315.1:374-823Ureaplasma urealyticum DNAfor urease subunits alpha, beta and gamma (EC 3.5.1.5), and its sequence number is X51315.1;
  • the target fragment of Chlamydia trachomatis is selected from the PmpF fragment, >CP035484.1:326414-326863 Chlamydia trachomatis strain tet9chromosome, complete genome, and its sequence number is CP035484.1;
  • the target fragment of HPV16 is selected from the L1 fragment, >MN542782.1:934-1383Human papillomavirus type 16isolate HPV16/Kp/L1/2019L1protein(L1)gene, complete cds, its sequence number is MN542782.1;
  • the target fragment of HPV18 is selected from the L1 fragment, > MH057749.1:1025-1474 Human papillomavirus type 18isolate HPJ18-7L1 (L1) gene, complete cds, its sequence number is MH057749.1;
  • the above fragments were connected to a plasmid vector for transformation, and 10 plasmids respectively containing the 10 target fragments were synthesized.
  • the plasmid construction process is as follows:
  • the plasmid vector used in the present invention is based on the commercialized plasmid pGH Vector (Shanghai Jierui Bioengineering Co., Ltd.), and the PCR product is inserted into the pGH Vector according to the conventional method recorded in "Molecular Cloning". , that is, construct 10 plasmids containing the 10 target fragments.
  • the plasmid can be stored in glycerol at -20°C for a long time, activated and extracted with plasmid DNA.
  • the plasmid DNA was extracted, and the concentration of the plasmid DNA was measured using the NanoDrop ND-2000 nucleic acid detector, and the copy number of the DNA was determined as a sensitivity standard to quantify the mother liquor.
  • the primers come from the conserved sequence of the target DNA, and tag sequences can be optionally added as needed, so that the size of multiplex PCR products can be easily distinguished by MALDI-TOF MS.
  • Embodiment 2 build the detection mass spectrometry model of 10 kinds of pathogens
  • a multiplex primer system for detecting 10 kinds of reproductive tract pathogens was used to construct a mass spectrometry model for the detection of 10 kinds of pathogens.
  • the components used in the kit for PCR, PCR product purification and single base extension are:
  • Reaction solution I dNTPs Tris-HCl, MgCl 2 323 ⁇ L/tube ⁇ 1 tube 2 Enzyme I Amplification enzyme, UNG enzyme 23 ⁇ L/tube ⁇ 1 tube 3 Amplification primer 10 pairs of PCR primers 115 ⁇ L/tube ⁇ 1 tube 4 Reaction solution II Tris-HCl, MgCl 2 173 ⁇ L/tube ⁇ 1 tube 5 Enzyme II SAP enzyme 58 ⁇ L/tube ⁇ 1 tube 6 Reaction solution III ddNTPs, Tris-HCl, MgCl 2 112 ⁇ L/tube ⁇ 1 tube 7 Enzyme III elongase 31 ⁇ L/tube ⁇ 1 tube 8 extension primer 10 extension primers 126 ⁇ L/tube ⁇ 1 tube 9 Quality Control Plasmid mix 50 ⁇ L/tube ⁇ 1 tube
  • the specific operation method is as follows:
  • PCR preparation area prepare 200ul PCR reaction tubes according to the number of samples to be tested (including positive quality control products, negative controls, and blank controls), and mark the sample number on the tube;
  • the PCR primer mixture and PCR reaction solution according to the ratio in the table below, put them in a centrifuge tube and mix well, add 4ul of the mixture to each PCR reaction tube for aliquoting. Due to factors such as residual pipette tips during the aliquoting process, it may not be enough to aliquot the required number of parts, it is recommended to appropriately enlarge the preparation volume of the mixture. For example, when there are 10 samples to be tested, the mixture can be prepared according to 10.5-11 samples.
  • the extension primer mixture and extension reaction solution according to the ratio in the table below, put them in a centrifuge tube and mix well. Due to factors such as residual pipette tips during the aliquoting process, it may not be enough to aliquot the required number of parts, it is recommended to appropriately enlarge the preparation volume of the mixture. For example, when there are 10 digestion products, the mixture can be prepared according to 10.5-11 samples.
  • Spotting Use a micropipette to draw 0.5ul of the purified product, and spot the sample on the target piece.
  • Miscellaneous peaks peaks that may appear in the non-reaction system are all miscellaneous peaks; peak type: no bifurcation, slant front, peak following, etc. are considered to be better peak types
  • Embodiment three isolate patient's pathogenic DNA
  • Sample source vaginal secretions. Sampling personnel should wear clean hands and wear disposable gloves before sampling, and should avoid the menstrual period of the subject when collecting.
  • the specimen collection and extraction methods are as follows: Cervical sampling swab: Rotate a plastic rod swab with a polypropylene fiber head and gently insert it into the vagina, stay for a while and then rotate clockwise 5 times. Take out the swab and dip it into a tube containing 3ml of sampling solution, discard the tail, and tighten the cap of the tube.
  • the swab preservation solution should not be stored for more than one week at 2-8°C, and should not be stored for more than one month at -20°C. It can be transported in a curler with ice or a foam box with ice. It is recommended to use fresh swab preservation solution as much as possible.
  • Bacterial DNA extraction kits were used to extract DNA from vaginal secretions. A total of 17 patients were extracted DNA to be tested.
  • Example 4 Using mass spectrometry model to detect pathogens in patients
  • Example 2 After performing PCR amplification, purification, and single base extension on the DNA to be tested in 17 patients, the target slices after spotting were detected and the results were judged by a Clin-TOF time-of-flight mass spectrometer.
  • the blank control was set as deionized water
  • the negative control was human genomic DNA
  • the positive control was a mixture of plasmid DNA fragments of 10 kinds of pathogens to be tested.
  • the detection peak values (m/z) are: 5919.8, 6363.2, 5997.9 respectively, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be detected are respectively: 5919.8, 6363.2, 5997.9, established according to Example 2
  • the mass spectrometry model so it is determined that the test results are: positive for Gardnerella vaginalis, positive for Candida albicans, and positive for Ureaplasma urealyticum.
  • the detection peak values (m/z) are: 6363.2, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be tested are: 6363.2, so the detection result is determined to be: Candida albicans positive.
  • the detection peak values (m/z) are: 6694.4, and the molecular weights (Da) of the single base extension products at the specific sites of the pathogen to be tested are respectively: 6694.4, so the detection result is determined to be: Mycoplasma genitalium positive.
  • the detection peak values (m/z) are: 5919.8, 6363.2, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be tested are: 5919.8, 6363.2, so the detection results are determined to be: Vaginal Gard Positive for narcotic bacteria and positive for candida albicans.
  • the detection peak values (m/z) are respectively: 5919.8, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be tested are respectively: 5919.8, so the detection result is determined to be: Gardnerella vaginalis positive.
  • the detection peak values (m/z) are: 8241.3, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be tested are respectively: 8241.3, so the detection result is determined to be: HPV16 positive.
  • the detection peak values (m/z) are: 6363.2, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be tested are respectively: 6363.2, so the detection result is determined to be positive for Candida albicans.
  • the detection peak values (m/z) are respectively: 6363.2, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be tested are respectively: 6363.2, so the detection result is determined to be positive for Candida albicans.
  • the detection peak values (m/z) are respectively: 5288.3, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be tested are respectively: 5288.3, so the detection result is determined to be Neisseria gonorrhoeae positive.
  • the detection peak values (m/z) are: 6363.2, 6077 respectively, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be tested are: 6363.2, 6077 respectively, so the detection results are determined to be: Candida albicans Positive, Staphylococcus aureus positive.
  • the detection peak values (m/z) are: 6077, and the molecular weight (Da) of the single-base extension product at the specific site of the pathogen to be tested is respectively: 6077, so the detection result is determined to be: Staphylococcus aureus positive.
  • the detection peak values (m/z) are: 5424.6, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be tested are respectively: 5424.6, so the detection result is determined to be: HPV18 positive.
  • the detection peak values (m/z) are: 5919.8, 6363.2, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be tested are: 5919.8, 6363.2, so the detection results are determined to be: Vaginal Gard Positive for narcotic bacteria and positive for candida albicans.
  • the detection peak values (m/z) are: 6986.6, and the molecular weight (Da) of the single-base extension product at the specific site of the pathogen to be tested is respectively: 6986.6, so the detection result is determined to be: Chlamydia trachomatis positive.
  • the detection peak values (m/z) are: 5447.6, 6363.2, respectively, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be tested are: 5447.6, 6363.2, so the detection results are determined to be: B chain Positive for coccus and positive for Candida albicans.
  • the detection peak values (m/z) are: 6694.4, and the molecular weight (Da) of the single-base extension product at the specific site of the pathogen to be tested is respectively: 6694.4, so the detection result is determined to be: positive for Mycoplasma genitalium.
  • the detection peak values (m/z) are: 6363.2, 6077, respectively, and the molecular weights (Da) of the single-base extension products at the specific sites of the pathogen to be tested are: 6363.2, 6077, so the detection results are determined to be: Candida albicans Positive, Staphylococcus aureus positive.
  • Figure 14-30 The baselines of the characteristic peaks in each figure are smooth, the signal-to-noise ratio is high, and the separation between adjacent signal peaks is high. It can be seen that the mass spectrometry method of the present invention can not only detect the products of multiple PCR at the same time, but also combine The multiplex PCR method quickly obtains the detection results, and avoids the disadvantages of long electrophoresis time and low resolution of small molecule fragments.

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Abstract

本发明提供了一种用于飞行时间质谱法检测8种生殖道病原菌及两种人乳头瘤病毒分型特定位点的引物组、产品及非诊断检测方法，所述生殖道病原菌包括金黄色葡萄球菌、B族链球菌、淋病奈瑟菌、阴道加德纳菌、白色念珠菌、生殖支原体、解脲支原体、沙眼衣原体以及HPV16、HPV18病毒分型。

Description

MALDI-TOF MS检测生殖道病原体的检测产品及用途 技术领域
本发明属于分子生物学检测领域，涉及一种利用激光飞行时间质谱技术快速鉴定10种生殖道病原体的的检测产品及用途。
背景技术
据世界卫生组织最新调查报告显示，对于成年女性而言，最常见的疾病不是感冒，而是生殖道感染，患病几率高达90％以上；最常见的不适症状不是发热、咳嗽、头痛等，而是阴部瘙痒、异味、疼痛，以及尿频，尿急等。而且女性许多其它疾病的发生也与生殖系统的功能异常密切相关，如骨质疏松、冠心病、糖尿病、黄褐斑等。从某种意义上讲，生殖系统的问题关乎女人一生的健康和幸福，也可以说是女人的“百病之源”。女性生殖道感染是发生在女性从外阴口至子宫这段腔道因细菌、病毒、寄生虫等病原微生物引起的感染性疾病，主要包括阴道炎(念珠菌性、滴虫性、老年性、细菌性阴道病)、淋病、宫颈炎(息肉、那氏囊肿)、盆腔炎等，是妇女的常见病，几乎每个成年女性和部分少女都曾有过。女性生殖道感染具有患病率高、无症状比例高、不就诊的比例高和得不到合理治疗比例高的特点，导致各种严重并发症和后遗症。女性生殖健康水平直接影响着人口素质，不容忽视。女性生殖道感染已经成为全球范围内危害严重的重要传染病之一。女性生殖感染相关因素由于女性生殖道因解剖、生理、性活动、分娩和卫生习惯等多种因素影响，易发生多种感染。
生殖道感染是妇科常见疾病，由细菌、病毒、白色假丝酵母菌、原虫、支原体、衣原体等多种病原体的侵袭所致，包括女性下生殖道的外阴炎、阴道炎、宫颈炎和上生殖道的盆腔炎性疾病。生殖道感染具流行范围广、复发率高、就诊率低的特点，是最常见的妇科疾病，国内患病率高达42.9％。生殖道感染若得不到及时诊断和正确治疗，可增加性传播疾病感染的风险，会引起异常子宫出血、不孕症、异位妊娠、慢性盆腔痛、流产、胎膜早破、早产、死胎、死产及先天性感染等并发症及后遗症，不仅危害妇女健康，还可危害胎儿及新生儿。生殖道感染已成为严重影响女性健康的社会公共卫生问题，给家庭及社会带来严重负担。
生殖道微生物菌群种类繁多，协调共生、相互制约，可以与宿主发生一系列复杂的相互作用，显著影响生殖道的生理机能和免疫功能，并在抵御机会性病原体的定植过程中发挥“第一道防线”作用。生殖道微生态环境的不平衡可导致女性趋向于发生各种妇科疾病，如阴道炎症、不孕不育、宫颈上皮内瘤变及宫颈癌等等。阴道炎可由一种病原体所致，单纯的病原体诊治相对简单，但我国阴道炎中有50％左右为混合性感染，利用生殖道微生态系统检测有利于精准诊治阴道混合性感染。近年来，随着女性不孕症的患病率逐年升高，不孕症越来越受到人们的关注。不孕症的发病机制复杂，其中女性生殖道微生物群落失衡与女性不孕不育密切相关。通过临床检测与评价女性阴道微生态平衡，对于女性不孕不育的诊治具有重要意义。高危型人乳头瘤病毒(HPV)持续感染是医学上公认的宫颈癌发生的主要原因和必要条件，其中HPV16和HPV18的感染与宫颈癌的发病密切相关。但感染HPV后仅5％-10％最终发展为宫颈癌前病变甚至宫颈癌。目前发现，生殖道微生态的不平衡可能是造成HPV持续感染继而发生宫颈病变的协同因素。重建及保护生殖道微生态平衡，尤其是维持乳杆菌为主、阴道正常的酸性环境，为HPV感染及相关宫颈疾病治疗提供研究的新方向。
生殖道病原体是人体重要的微生态体系之一，它们与宿主、环境之间构成了相互制约、相互协调的微生态平衡，这种动态平衡在维护女性生殖道健康中起到关键作用。女性生殖道病原体包括金黄色葡萄球、溶血性链球菌、淋病奈瑟菌、B族链球菌、大肠埃希菌、阴道加德纳菌、白色假丝酵母菌、支原体、沙眼衣原体和HPV等。
目前常用的病原体检测方法有实时荧光定量PCR法、PCR-毛细管电泳法、高通量测序法。但是，实时荧光定量PCR法检测通量低，难以对多种病原菌同时进行检测。PCR-毛细管电泳法虽然在自动化程度和分析时间上有了较大提升，但其检测通量远低于近年来新兴的分子生物学检测手段。高通量测序(NGS)能一次对几十万到几百万条DNA分子进行序列测定，在临床病原菌检测中发挥重要作用， 但是NGS技术对设备要求高、技术门槛高、平台构建需要大量资金投入，难以在临床应用领域进行推广。因此，临床应用领域迫切需要建立一种快速、准确、灵敏、高通量的生殖道病原体检测体系，为疾病的诊断、治疗及评估预后提供充足的依据。
基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry，简称MALDI-TOF MS)技术，是20世纪80年代末问世并迅速发展起来的一种质谱分析技术。其质量分析器是一个离子漂移管(ion dirft tube)，由离子源产生的离子首先被收集，在收集器中所有离子速度变为0，使用一个脉冲电场加速后进入无场漂移管，并以恒定速度飞向离子接收器，离子质量越大，到达接收器所用时间越长；离子质量越小，到达接收器所用时间越短。根据这一原理，可以把不同质量的离子按质荷比大小进行分离，准确检测多肽、蛋白质、核酸、多糖等生物大分子的分子质量和纯度，具有准确性高、灵活性强、通量大、检测周期短、性价比高的优点。
早期的MALDI-TOF MS质谱技术来检测临床微生物，主要是针对微生物的特征蛋白。林豪芸(“基质辅助激光解吸电离飞行时间质谱在临床微生物鉴定中的应用”，《中国人兽共患病学报》，2014年第8期)报道了“由于每种微生物都有其独特的蛋白质组成，与传统方法依靠微生物生理生化特性不同，蛋白质组成很少受到环境条件的影响，主要由遗传特性所决定，因此MALDI-TOF MS更加准确与可靠。在众多的方法之中，MALDI-TOF MS的一个主要优点是其鉴定微生物的普遍性。它可以同时分析大量目标且不要求选择特定的识别抗体或引物，而对发生基因变异的微生物，MALDI-TOF MS至少可以把他们鉴定到最近的邻居数据库中”。然而，该报道指出，虽然可以检测2-3种细菌的组分，“但当混合物中某一个物种数量占强烈主导地位时，MALDI-TOF MS的鉴定能力明显受限”。
中国专利申请CN102337223A，“产黄青霉抗真菌蛋白Pc-Arctin及其制备方法”，公开了一种检测产黄青霉抗真菌蛋白Pc-Arctin的MALDI-TOF鉴定方法，其中从平板上挑取产黄青霉A096孢子接种于SGY液体培养基培养，预处理得到粗蛋白溶液在色谱柱上分离纯化，并在羧甲基阳离子交换色谱柱上分离纯化，收集各洗脱组分，各组分离心超滤浓缩至所需体积，以宛氏拟青霉为敏感受试指示菌，追踪抗真菌活性组分，确定的活性成分判断获得蛋白的纯度；割取SDS-PAGE电泳图上的单一条带，进行MALDI-TOF鉴定。该方法仅适用于特定微生物，且需要多重蛋白纯化过程，最终用MALDI-TOF鉴定特征蛋白Pc-Arctin，其过程繁琐，适用面窄，不能实现质谱分类细菌或微生物的目的。
中国专利申请201110154723、“MALDI TOF MS辅助鉴定单增李斯特氏菌的方法”和201110154469、“MALDI TOF MS辅助鉴定霍乱弧菌的方法”公开了一种利用MALDI TOF MS技术辅助鉴定细菌的方法，包括：预处理细菌培养物，采集所有菌株样品的MALDI TOF MS图谱，根据软件制备细菌标准图谱，使用相同的方法检测并采集待测细菌的图谱，以及比较二者图谱，根据匹配分数进行判定。由于该方法使用常规的处理(通过无水乙醇、甲酸和乙腈处理，并辅以离心，最后吸取上清液进行检测)，尽管其在一定程度上能表征该细菌的特征图谱，但由于其待测物中含有蛋白质、脂类、脂多糖和脂寡糖、DNA、多肽及其它能被离子化的分子，其得到的图谱实质上是上述各种分子的图谱集合，因此既需要处理和比对的图谱信息量过大，并且因待检分子过于庞大而导致其图谱特征性偏低，只适用于某具体细菌而无法推广到其他大量的细菌检测中。
已有一些公开文献使用MALDI-TOF MS质谱技术对生殖道病原体进行分类和鉴定，例如，麻雅婷(“应用基质辅助激光解吸电离飞行时间质谱研究白色念珠菌血流感染的血清多肽指纹图谱”，《解放军医学杂志》，2018年1月1日，第43卷第1期)报道了通过建立白色念珠菌血流感染小鼠模型，应用MALDI-TOF MS联合弱阳离子交换磁珠(weak cation exchange，WCX)提取多肽技术，研究其血清多肽，并通过分析比较差异多肽峰建立相应的诊断模型，以辅助诊断真菌性的血流感染。虽然该诊断模型能够用于检测大肠埃希菌、金黄色葡萄球菌和白色念珠菌，但由于该方法需要从大量特征多肽片段中摸索出特征性曲线图峰值，并且各菌的多肽片段相互干扰，因此对于检测效果存在一定影响。
在检测临床微生物特征蛋白的基础上，近年来发展了MALDI-TOF MS检测临床微生物特征核酸片段的技术，该理论基础在于，组成遗传物质DNA的基本单元——四种核苷酸之间存在质量差异，如ddAMP、ddCMP、ddGMP、ddTMP的分子量依次为271.2Da、247.2Da、287.2Da、327.1Da(其中ddTMP 是经过修饰的)，它们之间的最小分子量差异在16Da，完全可以通过质谱进行分辨。使用质谱能够对碱基突变或多态位点(SNP)、插入/缺失(InDel)、甲基化位点、基因定量、拷贝数变化(copy number variation，CNV)等多种DNA变化类型进行检测。
中国专利申请201210272533.6“建立幽门螺杆菌核酸指纹图谱的方法及其产品”，公开了一种基于质谱技术快速鉴定幽门螺杆菌的方法，包括PCR扩增、SAP酶消化、转录和核酸酶切、纯化、质谱仪检测等步骤。该方法利用飞行时间质谱技术，对分子量和丰度各异的核酸片段进行检测，并形成谱图。但是，该方法中核酸片段经PCR扩增后，还需经过SAP酶消化、转录和核酸酶切，仅能识别单个碱基的变化，无法检测有特征序列的长片段DNA。
此外，基于MALDI-TOF MS，开发了一些核酸检测方法，如美国Agena公司的hME和iPLEX方法，德国Bruker公司的GOOD assay方法，韩国GeneMatrix公司的RFMP方法。各公司为了提高质谱仪的分辨率，对目标位点进行检测倾向于检测分子量较小的寡核苷酸片段，如RFMP方法通过对含单核苷酸多态性(SNP)位点的多重PCR产物进行限制性酶切，产生2000～4000Da左右的寡核苷酸片段进行检测，而GOOD assay方法通过磷酸二酯酶(Phosphodiesterase，PDE)将含SNP位点的寡核苷酸片段切割成1000～2000Da左右的小片段进行检测。然而，以上方法，都不可避免地存在操作复杂，耗时长等问题。
由于现有的利用MALDI-TOF MS检测临床病原体的技术，多数针对少数2-3种病原体进行检测，或是针对同一病原体的不同靶点或特定位点进行检测，因此目前缺少一种能同时对女性生殖道病原体，如金黄色葡萄球菌、B族链球菌、淋球菌、阴道加德纳菌、白色念珠菌、生殖支原体、解脲支原体、沙眼衣原体和人乳头瘤病毒(HPV16和HPV18)能快速、高效、准确的检测技术。
发明内容
基于上述技术存在的缺陷或不足，本发明原理之一在于：本发明提供了一种联合PCR技术、单碱基延伸技术和质谱检测技术，检测与待测病原体分型相关的特征片段的特征图谱。其中，针对10种病原体(金黄色葡萄球、B族链球菌、淋球菌、阴道加德纳菌、白色念珠菌、生殖支原体、解脲支原体、沙眼衣原体和HPV16和HPV18)，设计10种引物组，在多重PCR中同时扩增10种病原体的含有特定靶序列的DNA片段产物，并通过高效的单碱基引物对上述产物进行单碱基延伸，即延伸引物在10个特定位点处分别延伸一个核苷酸，使得所延伸的核苷酸类型，分别与该位点处的基因型相关。随后，对延伸产物进行纯化，通过MALDI TOF MS质谱对纯化后的待检混合物进行检测，通过质谱峰确定待检混合物中各物质分子量，并与预先计算的扩增产物的理论分子量进行比较，从而确定延伸产物中的待检测病原体类型。
本发明原理之二在于，为了解决多重PCR产物的相互干扰，以及排除不适宜的待检位点，本发明进行了优化筛选，从而确定了优化的扩增产物组合和延伸引物组合。
本发明原理之三在于，在多重PCR体系进一步优化过程中，为了使得延伸引物和延伸产物的分子量尽量分布在理想的质谱检测窗口(即4000～9000Da)，本发明在扩增引物中引入不影响PCR扩增的tag序列，使得最终的10种延伸产物既落入检测窗口，同时各自区分，避免了分子量的叠合，从而提高了检测准确率和灵敏度。
因此，本发明第一个目的是提供一种MALDI TOF-MS检测10种生殖道病原体特征片段引物组合物，其包括：
表1
Figure PCTCN2021143454-appb-000001
Figure PCTCN2021143454-appb-000002
其中，各位点对应的延伸引物及延伸产物分子量，以及所扩增片段的特征峰值如表2所示。
表2
Figure PCTCN2021143454-appb-000003
在一个实施方案中，上述PCR扩增引物序列为核心序列，其在5'端可包括保护碱基序列，优选5-15个 碱基。在一个具体实施方案中，保护碱基序列选自在5'段加入10bp的tag(ACGTTGGATG)，例如，PCR引物SEQ ID NO:1为5'-ACGTTGGATGGCCGTTATCTGTTTGTGATG-3’。在另一具体实施方案中，延伸引物的5'端也可以增加作为接头的碱基序列。
本发明第二个目的是提供了通过上述引物组合物所制备的检测产品，该检测产品可通过MALDI TOF MS来检测10种生殖道病原体检测相关的特征片段，其中，所述病原体的特征片段及对应的PCR扩增引物和延伸引物选自：
金黄色葡萄球的nuc片段及对应的PCR扩增引物为SEQ 1-2，延伸引物SEQ 21；
B族链球菌的scpB片段及对应的PCR扩增引物为SEQ 3-4，延伸引物SEQ 22；
淋病奈瑟菌的NG片段及对应的PCR扩增引物为SEQ 5-6，延伸引物SEQ 23；
阴道加德纳菌的16sRNA片段及对应的PCR扩增引物为SEQ 7-8，延伸引物SEQ 24；
白色念珠菌的ITS片段及对应的PCR扩增引物为SEQ 9-10，延伸引物SEQ 25；
生殖支原体的PA片段及对应的PCR扩增引物为SEQ 11-12，延伸引物SEQ 26；
解脲支原体的UreA片段及对应的PCR扩增引物为SEQ 13-14，延伸引物SEQ 27；
沙眼衣原体的PmpF片段及对应的PCR扩增引物为SEQ 15-16，延伸引物SEQ 28；
HPV16的L1片段及对应的PCR扩增引物为SEQ 17-18，延伸引物SEQ 29；
HPV18的L1片段及对应的PCR扩增引物为SEQ 19-20，延伸引物SEQ 30。
在一个实施方案中，该产品为检测试剂盒，包括：
(1)用于PCR扩增的反应液I，包括：PCR扩增引物，耐高温的DNA聚合酶，dNTPs，PCR反应缓冲液(包含dNTPs、Tris-HCl、MgCl 2)；
(2)用于PCR产物纯化的反应液II，其含有Tris-HCl、MgCl 2；
(3)用于单碱基延伸反应的反应液III，包括：延伸引物，耐高温的单碱基延伸酶，ddNTPs，延伸反应缓冲液，其含有Tris-HCl、MgCl 2。
在一个具体实施方案中，该试剂盒还可包括：阴性质控品，阳性质控品，纯化用树脂，点样及质谱检测用靶片，外切酶，人基因组DNA提取试剂等试剂。
上述实施方案中，其中在所述PCR扩增的反应体系中含有：
Figure PCTCN2021143454-appb-000004
在实施方案中，其中每组引物对的浓度控制在0.5-2μM之间。
其中在PCR扩增反应程序为：50℃预处理2min，然后在95℃预变性2min；95℃变性30s，56℃退火30s，72℃延伸30s，进行45个循环；72℃延伸5min。
其中SAP酶消化反应过程：向5ul扩增产物中加入1.5μL反应液II、0.5μL酶II，并在37℃下30min，65℃下5min各1个循环。
延伸反应过程：在消化产物中加入0.83μL反应液II、0.23μL酶II，0.94μL延伸引物，并在94℃下30s一个循环；94℃下5s，随后5个循环的56℃下5s和80℃下5s，共40个循环；最后在72℃下5分钟，共1个循环。
在上述任一实施方案中，其中延伸反应完毕后，在产物分析区向每PCR管/孔延伸产物中加入41μL超纯水和15mg树脂，颠倒混匀5分钟，5000rpm离心1min。
本发明第三个目的是使用上述引物组合物、检测产品来检测10种生殖道病原体的特征片段方法，包括如下步骤：
(1)多重PCR：使用特异性的PCR扩增引物，在一个反应体系中，对10种生殖道病原体的目的DNA区域同时进行扩增，得到含10处特定位点所在DNA区域的PCR产物；
(2)PCR产物纯化：对步骤(1)得到的PCR产物进行纯化，以减少对后续反应的干扰；
(3)单碱基延伸：使用10种特异性的延伸引物，在一个反应体系中，对步骤(2)得到的纯化后PCR产物进行多重单碱基延伸，延伸引物在对位病原体特定位点处伸一个核苷酸，该核苷酸与特定位点处的基因型互补配对；
(4)延伸产物纯化：对步骤(3)得到的延伸产物进行纯化，以获得高纯的延伸产物，避免盐离子等杂质对后续检测的影响；
(5)质谱仪检测：将步骤(4)得到的纯化产物点在含有基质的靶片上，放入质谱仪进行检测；
其中，所述病原体的特征片段及对应的PCR扩增引物和延伸引物选自：
金黄色葡萄球的nuc片段及对应的PCR扩增引物为SEQ 1-2，延伸引物SEQ 21；
B族链球菌的scpB片段及对应的PCR扩增引物为SEQ 3-4，延伸引物SEQ 22；
淋病奈瑟菌的NG片段及对应的PCR扩增引物为SEQ 5-6，延伸引物SEQ 23；
阴道加德纳菌的16sRNA片段及对应的PCR扩增引物为SEQ 7-8，延伸引物SEQ 24；
白色念珠菌的ITS片段及对应的PCR扩增引物为SEQ 9-10，延伸引物SEQ 25；
生殖支原体的PA片段及对应的PCR扩增引物为SEQ 11-12，延伸引物SEQ 26；
解脲支原体的UreA片段及对应的PCR扩增引物为SEQ 13-14，延伸引物SEQ 27；
沙眼衣原体的PmpF片段及对应的PCR扩增引物为SEQ 15-16，延伸引物SEQ 28；
HPV16的L1片段及对应的PCR扩增引物为SEQ 17-18，延伸引物SEQ 29；
HPV18的L1片段及对应的PCR扩增引物为SEQ 19-20，延伸引物SEQ 30。
在一个实施方案中，步骤2的纯化过程可以选自碱性磷酸酶消化、碱性磷酸酶和外切酶ExoI消化、切胶纯化、PCR纯化柱过柱等。在一个具体实施方案中，当使用碱性磷酸酶消化、或碱性磷酸酶和外切酶ExoI消化进行纯化后，进行高温酶失活处理。
在一个实施方案中，上述PCR引物序列为核心序列，其在5'端可包括保护碱基序列，优选5-15个碱基。在一个具体实施方案中，保护碱基序列选自在5'段加入10bp的tag(ACGTTGGATG)，例如，PCR引物SEQ ID NO:1为5'-ACGTTGGATGGCCGTTATCTGTTTGTGATG-3’。此外，延伸引物的5'端也可以增加作为接头的碱基序列。
在上述任一实施方案中，该方法可作为非诊断目的的应用，广泛用于对环境卫生、食品安全检测、进出口检验检疫等领域的病原体进行鉴定，以维护公共卫生安全。
本发明第四目的是提供一种用作上述质谱鉴定生殖道病原体的核酸指纹图谱库的制备方法，包括如下步骤：
(1)针对10种病原体的目的片段设计引物，并把这些片段连接到质粒载体上进行转化，合成了分别包含该10个目的片段的10个质粒，其中，
金黄色葡萄球菌的nuc目的片段的特异性引物组选自：SEQ1和SEQ2；
B族链球菌的scpB片段目的片段的特异性引物组选自：SEQ3和SEQ4；
淋病奈瑟菌的NG片段目的片段的特异性引物组选自：SEQ5和SEQ6；
阴道加德纳菌的16sRNA片段目的片段的特异性引物组选自：SEQ7和SEQ8；
白色念珠菌的ITS片段目的片段的特异性引物组选自：SEQ9和SEQ10；
生殖支原体的PA片段目的片段的特异性引物组选自：SEQ11和SEQ12；
解脲支原体的UreA片段目的片段的特异性引物组选自：SEQ13和SEQ14；
沙眼衣原体的PmpF片段目的片段的特异性引物组选自：SEQ15和SEQ16；
HPV16的L1片段目的片段的特异性引物组选自：SEQ17和SEQ18；
HPV18的L1片段目的片段的特异性引物组选自：SEQ19和SEQ20；
(2)提取10种质粒的混合DNA，并用步骤(1)中的引物组对上述混合DNA进行多重扩增，同时设置空白对照和阴性对照；
(3)对多重PCR产物进行纯化；
(4)加入单碱基延伸引物和延伸反应液，对扩增产物进行单碱基延伸，其中，
金黄色葡萄球菌的nuc目的片段的延伸引物选自：SEQ21；
B族链球菌的scpB片段目的片段的延伸引物选自：SEQ22；
淋病奈瑟菌的NG片段目的片段的延伸引物选自：SEQ23；
阴道加德纳菌的16sRNA片段目的片段的延伸引物选自：SEQ24；
白色念珠菌的ITS片段目的片段的延伸引物选自：SEQ25；
生殖支原体的PA片段目的片段的延伸引物选自：SEQ26；
解脲支原体的UreA片段目的片段的延伸引物选自：SEQ27；
沙眼衣原体的PmpF片段目的片段的延伸引物选自：SEQ28；
HPV16的L1片段目的片段的延伸引物选自：SEQ29；
HPV18的L1片段目的片段的延伸引物选自：SEQ30；
(5)将延伸引物产物直接与基质形成结晶混合物，点样于同一张芯片上；
(6)质谱仪检测，得到含有10种目的片段的特征核酸指纹图谱；
(7)将步骤(6)得到的核酸指纹特征图谱，通过计算机软件与空白对照、阴性对照、阳性对照进行比对分析，得到10种生殖道病原体的标准核酸指纹特征图谱。
在一个实施方案中，上述10种目的片段的核酸指纹特征图谱所对应的分型的特定片段分子量以及质谱特征峰值分别为：
金黄色葡萄球菌的nuc目的片段：6077(m/z)；
B族链球菌的scpB片段目的片段：5447.6(m/z)；
淋病奈瑟菌的NG片段目的片段：5288.3(m/z)；
阴道加德纳菌的16sRNA片段目的片段：5919.8(m/z)；
白色念珠菌的ITS片段目的片段：6363.2(m/z)；
生殖支原体的PA片段目的片段：6694.4(m/z)；
解脲支原体的UreA片段目的片段：5997.9(m/z)；
沙眼衣原体的PmpF片段目的片段：6986.6(m/z)；
HPV16的L1片段目的片段：8241.3(m/z)；
HPV18的L1片段目的片段：5424.6(m/z)。
在一个实施方案中，所述DNA吸附柱的固相支持物包括但不限于凝胶、树脂、硅土、硅胶、磁珠、玻璃粉、玻璃珠等。在一个具体实施方案中，所述基质为含有酸性成分的复合基质，该酸性成分包括但不限于甲酸、乙酸和柠檬酸。在另一个具体的实施方案中，所述芯片为飞行时间质谱专用微阵列芯片，其材质包括但不限于不锈钢、金刚石、单晶硅、石英晶体。
在另一实施方案中，步骤(2)中利用NanoDrop ND-2000核酸检测仪测量质粒DNA的浓度，确定DNA的拷贝数作为敏感度的标准品定量母液。
在其他实施方案中，上述PCR扩增引物序列为核心序列，其在5'端可包括保护碱基序列，优选5-15个碱基。在一个具体实施方案中，保护碱基序列选自在5'段加入10bp的tag(ACGTTGGATG)，例如，PCR引物SEQ ID NO:1为5'-ACGTTGGATGGCCGTTATCTGTTTGTGATG-3’。在另一具体实施方案中，延伸引物的5'端也可以增加作为接头的碱基序列。。
在一个具体实施方案中，所述质谱仪为MALDI TOF MS质谱仪。
在一个实施方案中，所述软件是发明人自行研究开发的BioExplore软件，其版权号为软著登字第136879号，登记号2009SR10700。
技术效果
与现有技术相比，本发明具有以下优点：
1、本发明首次提出利用多重PCR结合临床质谱实现对10种生殖道病原体相关特定片段的多重检测，具有极高的生物学价值。
2、敏感：本发明综合了多重PCR、单碱基延伸、质谱检测等技术为一体，既可通过PCR技术放大检测模板，又可通过质谱技术检测微量样本，综合了两种技术的优点，远远优于单独使用PCR检测病原体特定片段，因此它的检测灵敏度很高。
3、特异：单碱基延伸又称为“微测序”，使用特异性探针对核酸分子进行识别，具有测序技术的高准确性，特异性好、假阳性低等特点；特别的，不同于测序技术延伸数百个碱基，该技术仅延伸单个碱基，出错概率更低；
4、简便安全：操作简单、安全、自动化程度高、防污染；
5、本发明通过鉴定与病原体分型相关的特定位点的特征图谱，从而完成相关的病原体检测。本发明克服了以往技术一次性检测病原体类别过少的缺陷,成本低廉。
6、本发明所需的数据分析简单,只需观察谱图,无需复杂的生物信息学分析。
7、本发明成本低,无需荧光标记,降低了荧光化学探针加入导致的系统复杂性信号判读误差。
8、高自主化，使用自主研发的仪器、试剂、芯片和分析软件。
附图说明
见文末。
原理与定义
本发明提供了一种联合多重PCR、单碱基延伸和质谱检测等技术，检测10种生殖道病原体特定片段或位点的特征图谱，进而确定待检病原体类型的检测方案。
其原理在于：
在多重PCR步骤中，通过设计并使用合适的引物，从而能同时扩增10个特定位点所在DNA特定片段。
在单碱基延伸步骤中，对上一步多重PCR的产物依次进行纯化和多重单碱基延伸。其中，延伸引物共10条，分别与10个特定位点对应，并在对应的特定位点处延伸一个核苷酸，该核苷酸与特定位点处的基因型互补配对(如某特定位点处是A基因型，将在对应的延伸引物上延伸T核苷酸)。在单碱基延伸步骤中，采用ddNTP代替dNTP，因此，在延伸一个碱基后，延伸引物将终止延伸。
在质谱检测过程中，单碱基延伸产物在纯化后，点至含基质的靶片，并在真空环境中被激光激发，通过飞行管至检测器。不同物质通过飞行管的时间与它们的分子量呈负相关，即分子量越大，飞行速度越慢，到达检测器的时间越晚。
术语“保护碱基”，指在PCR引物的5'端额外增加的碱基。由于保护碱基的序列使得PCR引物(即核心引物)的分子量增大，可以避免反应剩余的PCR引物进入质谱检测窗口，以避免干扰检测效果。此外，延伸引物的5'端也可以适量增加碱基序列，但其作用并非如同PCR引物的保护碱基，使其超出检测窗口，而是适当调整延伸引物的分子量，使延伸引物及其产物在检测窗口内处于一个合理的位置。例如，当两个基因多态位点对应的延伸引物及产物的分子量接近时，通过给其中一个延伸引物增加碱基，改变引物及其产物的分子量，与其他延伸引物及产物的分子量之间拉大差距，以避免局部区域质谱峰过于集中而产生干扰和分辨不清，从而提高检测效果。因此，增加碱基后的延伸引物及产物的分子量，一定不会超出检测窗口。上述延伸引物的额外碱基可称为引物接头。
术语“碱性磷酸酶消化”，其作用是降解PCR反应后体系中残余dNTP，其原理是使dNTP的5'-P末端转换成5'-OH末端，从而失去与引物结合使引物延伸的能力，避免了对下一步单碱基延伸的影响。
术语“外切酶ExoI消化”，其作用是从单链DNA的一端开始按序催化水解组成DNA的dNTP之间3,5-磷酸二酯键，使单链DNA最终水解为dNTP。在本技术方案中用于降解PCR反应后残余的PCR引物。由于外切酶可以将单链的PCR引物切除，并不会在检测窗口中出现，因此使用 该外切酶时，所使用的PCR引物无需包括保护碱基。
术语“单碱基延伸”，又被称之为微测序(mini sequence)，指在体系中加入延伸引物和ddNTP，ddNTP与延伸引物的3'端连接形成延伸产物(即引物延伸了一个碱基)，根据碱基互补配对原则，由特定位点处基因型决定具体连接何种ddNTP，这个过程类似于PCR过程中dNTP根据互补链的碱基组成，逐个添加到PCR引物上。由于“ddNTP”与普通dNTP不同的是，在脱特定位点处连接一个ddNTP，而不能像PCR那样，不断的往下延伸，因此称之为单碱基延伸。单碱基延伸与测序过程非常相似，测序体系中加入的是dNTP和ddNTP的混合物，测序引物连接dNTP后将继续延伸，只有连接ddNTP后，方终止延伸，因此测序产生的是长短不一的核苷酸片段的混合物；单碱基延伸体系中加入只有ddNTP，延伸引物只能连接一个ddNTP，并终止延伸，因此单碱基延伸产生的是延伸引物仅延伸一个碱基的核苷酸片段。
术语“检测产品”，指用于检测特定位点基因型的任何常规产品，包括：检测试剂、检测芯片、检测载体，以及检测试剂盒等。
术语“ddNTP”是一种特殊的核苷酸，本技术方案共采用四种，它们之间存在分子量差异，如ddATP、ddCTP、ddGTP、ddTTP的分子量分别是271.2Da、247.2Da、287.2Da、327.1Da(其中ddTTP是修饰后的分子量)。当延伸引物根据特定位点的基因型而延伸不同的核苷酸，将形成分子量差异。通过质谱检测，可分辨出这种差异。例如，某特定位点若是A/G多态，对应的延伸引物长度为22个碱基(分子量7200Da)，当该位点处是A基因型，延伸引物将延伸一个T核苷酸并终止延伸，形成23个碱基长、分子量7487.2Da的延伸产物，当该位点处是G基因型，延伸引物将延伸一个C核苷酸并终止延伸，形成23个碱基长、分子量7447.2Da的延伸产物，两种产物之间存在40Da的分子量差异。即对该位点而言，若使用此7200Da的延伸引物，G基因型将对应7447.2Da的质谱峰，A基因型将对应7487.2Da的质谱峰。
术语“纯化”，指用于减少待检体系内其他物质对后续反应的影响的处理步骤。本发明的PCR产物纯化有两种方式：一是分离杂质并丢弃，二是使杂质失去活性。其中，切胶纯化、过纯化柱等都是通过电泳、纯化柱等分离杂质，并回收相对较纯的PCR产物，可以认为是第一种纯化方式，该方式一般耗时，操作复杂，特别是样本量大时；碱性磷酸酶的作用是降解(亦称“消化”)dNTP，使之不能继续作为DNA聚合酶或单碱基延伸酶的底物参与PCR或单碱基延伸反应，从而不干扰后续反应，可以认为是第二种纯化方式。应当指出的是，单独的外切酶ExoI不起纯化作用，当它与碱性磷酸酶混合使用时，其作用是预先将单链DNA(在反应完成后的PCR产物体系中，主要是剩余的PCR引物)降解成dNTP，再由碱性磷酸酶使dNTP继续降解。由于PCR引物被降解，不会进入最后的质谱检测步骤，因此，如果计划纯化步骤中增加ExoI外切酶处理，那么无需使用具有保护碱基的PCR引物。此外，在单碱基延伸步骤之前，由于外切酶和碱性磷酸酶都通过高温失活，其不会降解在单碱基延伸步骤中加入的单链的延伸引物、ddNTP等，因此避免对后续实验产生影响。
术语“m/z”质荷比是质量跟电荷的比，在谱图里面所得到的最大分子质量与最小电荷(即1个电荷)的比，也可以表示为分子量(Da，即相对分子量)。
术语“检测窗口”，指可用于质谱检测核苷酸分子量的范围，通常涉及引物的设计参考范围。其中，在设计延伸引物时，对于不同的特定位点，根据这些位点所在DNA区域的序列特点，以及特定位点的基因型，可以设计出分子量不同的延伸引物和延伸产物，避免不同延伸引物及产物之间由于分子量接近而存在干扰，从而可在一个相对宽阔的检测窗口，如4000-9000Da，实现对多个特定位点的检测。
应当指出的是，鉴于以上核酸质谱的特殊性，例如，其需先通过PCR反应扩增出含特定位点的片段，然后通过延伸引物延伸出特定位点的碱基；各特定位点的PCR反应、延伸反应间无明显干扰；各特定位点的延伸引物、产物间分子量要有足够大的差异以便实现区分等，因此，并非所有的位点均可以用来进行核酸质谱的检测，也并不是所有针对特定位点设计的引物均可 用以进行多重PCR反应和多重单碱基延伸反应。例如，Cláudia M.B等人(Optimization of a multiplex minisequencing protocol for population studies and medical genetics，Genet.Mol.Res 4(2005)115-125)指出，在进行多重PCR反应前需首先对单重PCR的反应效果进行验证，如果单重PCR扩增效率低则需要放弃，另外，若PCR产物长度偏长，多重PCR效果会比较差，也需要放弃。
此外，在核酸质谱检测过程中，多重扩增过程的干扰作用，对于最终获得的延伸产物也存在影响。Sascha Sauer等人(Typing of single nucleotide polymorphisms by MALDI mass spectrometry:Principles and diagnostic applications，Clinica Chimica Acta 363(2006)95–105)和Heyi Yang等人(Multiplex single-nucleotide polymorphism genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry，Analytical Biochemistry 314(2003)54–62)在利用MALDI质谱技术研究核酸质谱检测过程中提出，所设计的多重引物应具有相近的熔解温度(Tm值)并彼此间的相互作用力较弱。如果引物之间的相互作用力过于强烈(ΔG的最小值为-10kcal/mol)，那么必须放弃该理论设计的引物而重新进行设计；当同一反应体系中存在多重反应引物，那么多重扩增的规模主要受限于引物之间的相互作用程度，从而影响核酸质谱检测过程；此外，为了准确区分不同碱基之间的差异，特别是腺嘌呤(A)和胸腺嘧啶(T)(4种碱基中这二者分子量之间差值最小，为9Da)，要求的寡核苷酸长度一般不超过40个碱基，实际应用中，质谱检测窗口的分子量范围一般为4000～9000Da，即要求所涉及的延伸引物和产物的分子量尽量分布在4000～9000范围之内。同时，要避免各延伸引物及其延伸产物之间的叠合。由此可见，并非所有特定位点均可应用于核酸质谱尤其是多重核酸质谱的检测，其实际效果会受到多种实验因素的影响，因此需要通过实验来验证特定位点的可行性以及筛选不同引物的组合。
附图说明：
图1-图13：实施例二中对待测病原体DNA进行核酸质谱检测的各组空白对照、阴性对照、阳性对照和待 测样品的质谱结果图，其中箭头所示为，
图1为金黄色葡萄球菌的nuc目的片段(6077Da)所对应的特征峰：6077m/z；图2为B族链球菌的scpB片段目的片段(5447.6Da)所对应的特征峰：5447.6m/z；图3为淋病奈瑟菌的NG片段目的片段(5288.3Da)所对应的特征峰：5288.3m/z；图4为阴道加德纳菌的16sRNA片段目的片段(5919.8Da)所对应的特征峰：5919.8m/z；图5为白色念珠菌的ITS片段目的片段(6363.2Da)所对应的特征峰：6363.2m/z；图6为生殖支原体的PA片段目的片段(6694.4Da)所对应的特征峰：6694.4m/z；图7为解脲支原体的UreA片段目的片段(5997.9Da)所对应的特征峰：5997.9m/z；图8为沙眼衣原体的PmpF片段目的片段(6986.6Da)所对应的特征峰：6986.6m/z；图9为HPV16的L1片段目的片段(8241.3Da)所对应的特征峰：8241.3m/z；图10为HPV18的L1片段目的片段(5424.6Da)所对应的特征峰：5424.6m/z；图11为阳性对照，从左至右分别为：淋病奈瑟菌NG片段(5288.3Da)所对应的特征峰：5288.3m/z；HPV18L1片段(5424.6Da)所对应的特征峰：5424.6m/z；B族链球菌scpB片段(5447.6Da)所对应的特征峰：5447.6m/z；阴道加德纳菌16sRNA片段(5919.8Da)所对应的特征峰：5919.8m/z；解脲支原体UreA片段(5997.9Da)所对应的特征峰：5997.9m/z；金黄色葡萄球菌nuc目的片段(6077Da)所对应的特征峰：6077m/z；白色念珠菌ITS片段(6363.2Da)所对应的特征峰：6363.2m/z；生殖支原体PA片段(6694.4Da)所对应的特征峰：6694.4m/z；沙眼衣原体PmpF片段(6986.6Da)所对应的特征峰：6986.6m/z；HPV16L1片段(8241.3Da)所对应的特征峰：8241.3m/z；图12为阴性对照，谱图均为延伸引物出峰位置，无阳性靶标特征峰(延伸产物峰)，即未检出任何阳性病原体；图13为空白对照，谱图均为延伸引物出峰位置，无阳性靶标特征峰(延伸产物峰)，即未检出任何阳性病原体；
图14-图30：实施例四中对患者病原体DNA的质谱结果图，其中箭头所示，图14N1，检测峰值(m/z)分别是：5919.8，6363.2，5997.9；图15N2，检测峰值(m/z)分别是：6363.2；
图16N3，检测峰值(m/z)分别是：6694.4；图17N4，检测峰值(m/z)分别是：5919.8，6363.2；图18 N5，检测峰值(m/z)分别是：5919.8；图19N6，检测峰值(m/z)分别是：8241.3；图20N7，检测峰值(m/z)分别是：6363.2；图21N8，检测峰值(m/z)分别是：6363.2；图22N9，检测峰值(m/z)分别是：5288.3；图23N10，检测峰值(m/z)分别是：6363.2，6077；图24N11，检测峰值(m/z)分别是：6077；图25N12，检测峰值(m/z)分别是：5424.6；图26N13，检测峰值(m/z)分别是：5919.8，6363.2；图27N14，检测峰值(m/z)分别是：6986.6；图28N15，检测峰值(m/z)分别是：5447.6，6363.2；图29N16，检测峰值(m/z)分别是：6694.4；图30N17，检测峰值(m/z)分别是：6363.2，6077；
图31-图33：实施例四中空白对照、阴性对照、阳性对照质谱结果图。其中，图31空白对照，谱图均为延伸引物出峰位置，无阳性靶标特征峰(延伸产物峰)，即未检出任何阳性病原体；图32阴性对照，谱图均为延伸引物出峰位置，无阳性靶标特征峰(延伸产物峰)，即未检出任何阳性病原体；图33阳性对照从左至右分别为：淋病奈瑟菌NG片段(5288.3Da)所对应的特征峰：5288.3m/z；HPV18L1片段(5424.6Da)所对应的特征峰：5424.6m/z；B族链球菌scpB片段(5447.6Da)所对应的特征峰：5447.6m/z；阴道加德纳菌16sRNA片段(5919.8Da)所对应的特征峰：5919.8m/z；解脲支原体UreA片段(5997.9Da)所对应的特征峰：5997.9m/z；金黄色葡萄球菌nuc目的片段(6077Da)所对应的特征峰：6077m/z；白色念珠菌ITS片段(6363.2Da)所对应的特征峰：6363.2m/z；生殖支原体PA片段(6694.4Da)所对应的特征峰：6694.4m/z；沙眼衣原体PmpF片段(6986.6Da)所对应的特征峰：6986.6m/z；HPV16L1片段(8241.3Da)所对应的特征峰：8241.3m/z。
具体实施方式
下面结合具体实施例，进一步阐述本发明。下列实施例中未注明具体条件的实验方法，通常按照常规条件实施。
需特别指出的是，尽管本发明的实施例中呈现的是50,000Da以下多重PCR产物的质谱检测数据，但50,000-100,000Da之间的多重PCR产物也可检测到，因此应用本发明对50,000-100,000Da之间的多重PCR产物质谱检测也包括在本发明所要求的权利范围之内。
实施例一、引物设计
本发明中所有检测目标区域都是单区域检测，PCR引物，与上述各参考PCR引物的3'端对应，保证能扩增，5'端由Primer5引物设计软件，根据扩增效率和多重PCR兼容性进行了调整，不影响引物的特异性，不会增加引物的非特异性。
针对10种病原体的目的检测区段(金黄色葡萄球菌的nuc片段、B族链球菌的scpB片段等)设计引物，经过检索NCBI数据库，各病原体目的片段来源如下：
金黄色葡萄球菌的目的片段：金黄色葡萄球菌的目的片段选自nuc片段，>DQ399678.1:1-450 Staphylococcus aureus nuclease(nuc)gene,partial cds，其序列号为DQ399678.1；
B族链球菌的目的片段：B族链球菌的目的片段选自scpB片段：>CP053027.1:1577249-1577698 Streptococcus agalactiae strain 01173chromosome,complete genome，其序列号为CP053027.1；
淋病奈瑟菌的目的片段：淋病奈瑟菌的目的片段选自NG片段，>AP023076.1:1148-1597Neisseria gonorrhoeae TUM16691plasmid pMTY16691DNA,complete genome，其序列号为AP023076.1；
阴道加德纳菌的目的片段：阴道加德纳菌的目的片段选自16sRNA片段，>L08167.1Gardnerella vaginalis 3'end of 16S ribosomal RNA,internal transcribed spacer,and 5'end of 23S ribosomal RNA，其序列号为L08167.1；
白色念珠菌的目的片段：白色念珠菌的目的片段选自ITS片段，>MK805514.1:77-526Candida albicans strain TBS611small subunit ribosomal RNA gene,partial sequence；internal transcribed spacer 1,5.8S ribosomal RNA gene,and internal transcribed spacer 2,complete sequence；and large subunit ribosomal RNAgene,partial sequence，其序列号为MK805514.1；
生殖支原体的目的片段：生殖支原体的目的片段选自PA片段，>KP318805.1:413-862Mycoplasma genitalium strain Seattle1MgpB adhesin gene,complete cds，其序列号为KP318805.1；
解脲支原体的目的片段：解脲支原体的目的片段选自UreA片段，>X51315.1:374-823Ureaplasma  urealyticum DNAfor urease subunits alpha,beta and gamma(EC 3.5.1.5)，其序列号为X51315.1；
沙眼衣原体的目的片段：沙眼衣原体的目的片段选自PmpF片段，>CP035484.1:326414-326863 Chlamydia trachomatis strain tet9chromosome,complete genome，其序列号为CP035484.1；
HPV16的目的片段：HPV16的目的片段选自L1片段，>MN542782.1:934-1383Human papillomavirus type 16isolate HPV16/Kp/L1/2019L1protein(L1)gene,complete cds，其序列号为MN542782.1；
HPV18的目的片段：HPV18的目的片段选自L1片段，>MH057749.1:1025-1474Human papillomavirus type 18isolate HPJ18-7L1(L1)gene,complete cds，其序列号为MH057749.1；
将以上这些片段连接到质粒载体上进行转化，合成了分别包含该10个目的片段的10个质粒。
质粒构建过程如下所述：本发明中所用的质粒载体为在商品化质粒pGH Vector(上海捷瑞生物工程有限公司)的基础上，根据《分子克隆》记载的常规方法，将PCR产物插入pGH Vector，即构建包含该10个目的片段的10个质粒。所述质粒可长期保存于-20℃甘油中，用时活化并提取质粒DNA。
经鉴定后提取质粒DNA，利用NanoDrop ND-2000核酸检测仪测量质粒DNA的浓度，确定DNA的拷贝数作为敏感度的标准品定量母液。
引物来自于目标DNA的保守序列，可选地根据需要加入tag序列，使多重PCR产物的大小能够容易地由MALDI-TOF MS进行区分。
每个质粒及其对应的特异性PCR引物信息如表1，由上海捷瑞生物工程有限公司合成。
表1.引物信息表
Figure PCTCN2021143454-appb-000005
Figure PCTCN2021143454-appb-000006
实施例二、构建10种病原体的检测质谱模型
使用ABI 9700型PCR仪，按说明书对10种生殖道病原体相关的特定位点进行检验。
本实施例使用检测10种生殖道病原体的多重引物体系，构建10种病原体的检测质谱模型。设置空白对照为去离子水、阴性对照为人类基因组DNA，阳性对照为检测10种生殖道病原体的各靶标的质粒混合物。
试剂盒中用于PCR、PCR产物纯化和单碱基延伸的组分为：
序号 组分名称 主要成分 包装规格
1 反应液I dNTPs、Tris-HCl、MgCl 2 323μL/管×1管
2 酶I 扩增酶、UNG酶 23μL/管×1管
3 扩增引物 10对PCR引物 115μL/管×1管
4 反应液II Tris-HCl、MgCl 2 173μL/管×1管
5 酶II SAP酶 58μL/管×1管
6 反应液III ddNTPs、Tris-HCl、MgCl 2 112μL/管×1管
7 酶III 延伸酶 31μL/管×1管
8 延伸引物 10条延伸引物 126μL/管×1管
9 质控品 质粒混合物 50μL/管×1管
按说明书，具体操作方法如下：
1.PCR扩增
1.1在PCR配液区，按照待检样品数(含阳性质控品、阴性对照、空白对照)准备200ul PCR反应管，并在管上标记样本编号；
1.2从试剂盒中取出PCR引物混合液、PCR反应液，使其自然解冻，涡旋振荡使其充分混匀，瞬时离心至管底；
1.3根据样本数目，按下表的比例取出PCR引物混合液和PCR反应液，置于一个离心管中混匀，按每PCR反应管加入4ul混合物进行分装。由于分装过程中，移液器吸头残留等因素可能造成不足以分装出所需的份数，建议适当放大混合物的配制体积。例如有10份待测样品时，可按10.5-11份样品配制混合物。
组分名称 单反应体积取用量(μL)
反应液I 2.8
酶I 0.2
扩增引物* 1.0
合计 4
1.4在PCR扩增区内向每管混合物中加入1ul待测样品，使每份PCR反应体系总体积为5ul。其中，阴性对照为人类基因组DNA，阳性对照为质粒混合物，空白对照为去离子水。
1.5将PCR反应管置于PCR扩增仪中，按下表的程序进行PCR扩增反应。
Figure PCTCN2021143454-appb-000007
2.SAP酶消化
2.1向PCR产物中加入2ul酶切反应液，然后将PCR反应管置于PCR扩增仪中，执行下表程序。
组分名称 单反应体积(μL)
反应液II 1.5
酶II 0.5
合计 2.0
2.2将SAP反应管置于PCR扩增仪中，按下表的程序进行SAP消化反应。
温度(℃) 时间 循环数
37 30min 1
65 5min 1
4 Hold  
3.延伸
3.1在PCR配液区，根据样本数目，按下表的比例取出延伸引物混合液和延伸反应液，置于一个离心管中混匀。由于分装过程中，移液器吸头残留等因素可能造成不足以分装出所需的份数，建议适当放大混合物的配制体积。例如有10份酶切产物时，可按10.5-11份样品配制混合物。
组分名称 单反应体积(μL)
反应液III 0.83
酶III 0.23
延伸引物 0.94
合计 2.00
3.2在PCR扩增区，按每管酶切产物加入2ul混合物进行分装。
3.3将PCR反应管置于PCR扩增仪中，按下表的程序进行延伸反应。
Figure PCTCN2021143454-appb-000008
4.纯化：在PCR扩增区向每管延伸产物中加入41μL超纯水和15mg树脂，颠倒混匀5分钟。
5.点样：使用微量移液器，吸取0.5ul纯化产物，点样至靶片。
6.上机检测及结果判读：将上述纯化产物通过临床飞行时间质谱Clin-TOF-Ⅱ(毅新博创生物科技有限公司生产的MALDI-TOF MS)质谱仪进行检测。
7.结果判读：使用发明人研制的Clin-TOF型飞行时间质谱仪对点样后的靶片进行检测和结果判断。
8.检测质量标准
Figure PCTCN2021143454-appb-000009
杂峰：非本反应体系中可能出现的峰均为杂峰；峰型：无分叉，偏锋，跟峰等情况视为峰型较好
8.检测结果
将所述单碱基延伸产物均点样至同样一张芯片上，进行核酸质谱检测。
阳性靶标所对应的目的片段分子量所代表的特征峰(m/z)：金黄色葡萄球菌的nuc目的片段(6077Da)所对应的特征峰：6077m/z；B族链球菌的scpB片段目的片段(5447.6Da)所对应的特征峰：5447.6m/z；淋病奈瑟菌的NG片段目的片段(5288.3Da)所对应的特征峰：5288.3m/z；阴道加德纳菌的16sRNA片段目的片段(5919.8Da)所对应的特征峰：5919.8m/z；白色念珠菌的ITS片段目的片段(6363.2Da)所对应的特征峰：6363.2m/z；生殖支原体的PA片段目的片段(6694.4Da)所对应的特征峰：6694.4m/z；解脲支原体的UreA片段目的片段(5997.9Da)所对应的特征峰：5997.9m/z；沙眼衣原体的PmpF片段目的片段(6986.6Da)所对应的特征峰：6986.6m/z；HPV16的L1片段目的片段(8241.3Da)所对应的特征峰：8241.3m/z；HPV18的L1片段目的片段(5424.6Da)所对应的特征峰：5424.6m/z；将得到的核酸指纹特征图谱，通过软件与空白对照、阴性对照、阳性对照进行比对分析，结果显示：(1)空白对照：无上述阳性靶标特征峰，即未检出任何阳性病原体；(2)阴性对照：无上述阳性靶标特征峰，即未检出任阳性何病原体；(3)阳性对照：所有目的片段的阳性特征峰均正确检出，延伸效率均为100％；此外，所有组均无明显杂峰，基线平稳；谱图中3000-10000Da范围内无杂峰或<2根杂峰。因此，构建出可以检出10种生殖道病原体的质谱特征模型。质谱检测结果如图1-13。
实施例三、分离患者病原体DNA
样本来源：阴道分泌物。采样人员采样前应穿上洗净双手并带上一次性手套，采集时应避开受检者的经期。标本采集及提取方法如下：宫颈采样拭子：将1根聚丙烯纤维头的塑料杆拭子转动着轻轻插入阴道，停留片刻后顺时针旋转5次。取出拭子浸入含3ml采样液的管中，尾部弃去，旋紧管盖。拭子保存液在2-8℃保存不应超过一周，-20℃保存不应超过一个月，并可采用冰壶加冰或泡沫箱加冰密封进行运输，建议尽量采用新鲜拭子保存液。
样本提取：采用细菌DNA提取试剂盒对阴道分泌物进行DNA提取。共提取17例患者的待测DNA。
实施例四、利用质谱模型检测患者病原体
根据实施例二的方法，对17例患者的待测DNA进行PCR扩增、纯化、单碱基延伸后，通过Clin-TOF型飞行时间质谱仪对点样后的靶片进行检测和结果判断。同时按照实施例2，设置空白对照为去离子水、阴性对照为人类基因组DNA，阳性对照为10种病原体待检片段质粒DNA混合物。
质谱结果如图14-30所示：
样品N1，检测峰值(m/z)分别是：5919.8，6363.2，5997.9，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：5919.8，6363.2，5997.9，根据实施例2建立的质谱模型，因此确定检测结果为：阴道加德纳菌阳性，白色念珠菌阳性，解脲支原体阳性。样品N2，检测峰值(m/z)分别是：6363.2，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：6363.2，因此确定检测结果为：白色念珠菌阳性。样品N3，检测峰值(m/z)分别是：6694.4，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：6694.4，因此确定检测结果为：生殖道支原体阳性。样品N4，检测峰值(m/z)分别是：5919.8，6363.2，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：5919.8，6363.2，因此确定检测结果为：阴道加德纳菌阳性，白色念珠菌阳性。样品N5，检测峰值(m/z)分别是：5919.8，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：5919.8，因此确定检测结果为：阴道加德纳菌阳性。
样品N6，检测峰值(m/z)分别是：8241.3，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：8241.3，因此确定检测结果为：HPV16阳性。样品N7，检测峰值(m/z)分别是：6363.2，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：6363.2，因此确定检测结果为：白色念珠菌阳性。样品N8，检测峰值(m/z)分别是：6363.2，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：6363.2，因此确定检测结果为：白色念珠菌阳性。样品N9，检测峰值(m/z)分别是：5288.3，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：5288.3，因此确定检测结果为：淋病奈瑟菌阳性。样品N10，检测峰值(m/z)分别是：6363.2，6077，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：6363.2，6077，因此确定检测结果为：白色念珠菌阳性，金黄色葡萄球菌阳性。样品N11，检测峰值(m/z)分别是：6077，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：6077，因此确定检测结果为：金黄色葡萄球菌阳性。
样品N12，检测峰值(m/z)分别是：5424.6，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：5424.6，因此确定检测结果为：HPV18阳性。样品N13，检测峰值(m/z)分别是：5919.8，6363.2，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：5919.8，6363.2，因此确定检测结果为：阴道加德纳菌阳性，白色念珠菌阳性。样品N14，检测峰值(m/z)分别是：6986.6，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：6986.6，因此确定检测结果为：沙眼衣原体阳性。样品N15，检测峰值(m/z)分别是：5447.6，6363.2，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：5447.6，6363.2，因此确定检测结果为：B族链球菌阳性，白色念珠菌阳性。样品N16，检测峰值(m/z)分别是：6694.4，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：6694.4，因此确定检测结果为：生殖道支原体阳性。样品N17，检测峰值(m/z)分别是：6363.2，6077，其中待检病原体特定位点的单碱基延伸产物分子量(Da)分别是：6363.2，6077，因此确定检测结果为：白色念珠菌阳性，金黄色葡萄球菌阳性。
图14-30：各图中目的特征峰基线平滑，信噪比高，相邻信号峰之间分离度高，由此可见，本发明质谱法既能对多重PCR的产物同时进行检测，又能联合多重PCR方法快速获得检测结果，而避免了小分子片段电泳时间过长、分辨率低的缺点。

Claims (10)

  1. 一种MALDI TOF-MS检测10种生殖道病原体特征片段的检测产品，其中该检测产品包含以下引物组合物：
    金黄色葡萄球的nuc片段及对应的PCR扩增引物为SEQ 1-2，延伸引物SEQ 21；
    B族链球菌的scpB片段及对应的PCR扩增引物为SEQ 3-4，延伸引物SEQ 22；
    淋病奈瑟菌的NG片段及对应的PCR扩增引物为SEQ 5-6，延伸引物SEQ 23；
    阴道加德纳菌的16sRNA片段及对应的PCR扩增引物为SEQ 7-8，延伸引物SEQ 24；
    白色念珠菌的ITS片段及对应的PCR扩增引物为SEQ 9-10，延伸引物SEQ 25；
    生殖支原体的PA片段及对应的PCR扩增引物为SEQ 11-12，延伸引物SEQ 26；
    解脲支原体的UreA片段及对应的PCR扩增引物为SEQ 13-14，延伸引物SEQ 27；
    沙眼衣原体的PmpF片段及对应的PCR扩增引物为SEQ 15-16，延伸引物SEQ 28；
    HPV16的L1片段及对应的PCR扩增引物为SEQ 17-18，延伸引物SEQ 29；
    HPV18的L1片段及对应的PCR扩增引物为SEQ 19-20，延伸引物SEQ 30。
  2. 权利要求1的检测产品，其中各片段对应的延伸产物分子量以及所扩增片段的特征峰值如下
    Figure PCTCN2021143454-appb-100001
  3. 权利要求1或2的检测产品，其中该检测产品为检测试剂盒，包括：
    (1)用于PCR扩增的反应液I，包括：PCR扩增引物，耐高温的DNA聚合酶，dNTPs，PCR反应缓冲液(包含dNTPs、Tris-HCl、MgCl 2)；
    (2)用于PCR产物纯化的反应液II，其含有Tris-HCl、MgCl 2；
    (3)用于单碱基延伸反应的反应液III，包括：延伸引物，耐高温的单碱基延伸酶，ddNTPs，延伸反应缓冲液，其含有Tris-HCl、MgCl 2。
  4. 权利要求4的检测产品，该检测试剂盒还可包括：阴性质控品，阳性质控品，纯化用树脂，点样及质谱检测用靶片，外切酶，人基因组DNA提取试剂等试剂。
  5. 权利要求5的检测产品，其中在所述PCR扩增的反应体系中含有：
    Figure PCTCN2021143454-appb-100002
    在实施方案中，其中每组引物对的浓度控制在0.5-2μM之间。
  6. 通过权利要求1-5任一所述的检测产品，检测10种生殖道病原体的特征片段方法，包括：
    (1)多重PCR：使用特异性的PCR扩增引物，在一个反应体系中，对10种生殖道病原体的目的DNA区域同时进行扩增，得到含10处特定位点所在DNA区域的PCR产物；
    (2)PCR产物纯化：对步骤(1)得到的PCR产物进行纯化，以减少对后续反应的干扰；
    (3)单碱基延伸：使用10种特异性的延伸引物，在一个反应体系中，对步骤(2)得到的纯化后PCR产物进行多重单碱基延伸，延伸引物在对位病原体特定位点处伸一个核苷酸，该核苷酸与特定位点处的基因型互补配对；
    (4)延伸产物纯化：对步骤(3)得到的延伸产物进行纯化，以获得高纯的延伸产物，避免盐离子等杂质对后续检测的影响；
    (5)质谱仪检测：将步骤(4)得到的纯化产物点在含有基质的靶片上，放入质谱仪进行检测；
    其中，所述病原体的特征片段及对应的PCR扩增引物和延伸引物选自：
    金黄色葡萄球的nuc片段及对应的PCR扩增引物为SEQ 1-2，延伸引物SEQ 21；
    B族链球菌的scpB片段及对应的PCR扩增引物为SEQ 3-4，延伸引物SEQ 22；
    淋病奈瑟菌的NG片段及对应的PCR扩增引物为SEQ 5-6，延伸引物SEQ 23；
    阴道加德纳菌的16sRNA片段及对应的PCR扩增引物为SEQ 7-8，延伸引物SEQ 24；
    白色念珠菌的ITS片段及对应的PCR扩增引物为SEQ 9-10，延伸引物SEQ 25；
    生殖支原体的PA片段及对应的PCR扩增引物为SEQ 11-12，延伸引物SEQ 26；
    解脲支原体的UreA片段及对应的PCR扩增引物为SEQ 13-14，延伸引物SEQ 27；
    沙眼衣原体的PmpF片段及对应的PCR扩增引物为SEQ 15-16，延伸引物SEQ 28；
    HPV16的L1片段及对应的PCR扩增引物为SEQ 17-18，延伸引物SEQ 29；
    HPV18的L1片段及对应的PCR扩增引物为SEQ 19-20，延伸引物SEQ 30。
  7. 权利要求6的方法，其中步骤2的纯化过程可以选自碱性磷酸酶消化、碱性磷酸酶和外切酶ExoI消化、切胶纯化、PCR纯化柱过柱等。
  8. 权利要求7的方法，当使用碱性磷酸酶消化、或碱性磷酸酶和外切酶ExoI消化进行纯化后，进行高温酶失活处理。
  9. 权利要求6-8任一所述的方法，其中PCR扩增引物序列为核心序列，其在5'端可包括保护碱基序列，所述保护碱基序列选自在5'段加入10bp的tag：ACGTTGGATG。
  10. 权利要求6-8任一所述的方法，其中该方法可作为非诊断目的的应用，用于对环境卫生、食品安全检测、进出口检验检疫等领域的病原体进行鉴定。
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