Probe, probe mixture and method for performing bacterial typing by using probe mixture

Abstract

The invention discloses a probe, a probe mixture and a method for performing bacterial typing by using the probe mixture. The nucleic acid sequence of the probe comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 5-17. The probe provided by the invention can be used for remarkably distinguishing positive and negative samples and improving the detection specificity, and according to a bacterial typing method provided by the invention, trace blood or tissue cells can be directly used as a PCR amplification template, a sequence for judging whether organs are infected with pathogenic bacteria or not can be successfully amplified within 40 minutes, and infection type identification can be completed within 100 minutes.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a probe, a probe mixture and a method for bacterial typing using the same. Background Art

[0002] In the field of organ transplantation, donor acquisition is the most important factor in whether an organ transplant can be carried out. With the improvement of relevant domestic laws and regulations and the popularization and advocacy of organ donation, more and more people choose to donate their organs free of charge after death. This key factor is gradually being resolved. However, how to efficiently use these hard-won donor organs is becoming a problem that people are paying more and more attention to. After the organ is separated from the donor, it can only maintain certain physiological functions for a short time under appropriate storage conditions. The donor organ needs to be implanted into the recipient as soon as possible. In order to ensure that the donor organ can survive in the recipient for a long time, it is necessary to screen the donor organ for pathogen infection. For donors in good physical condition, pathogen infection is often a "hidden" infection, and the donor presents non-characteristic infection symptoms, but it is a fatal infection for recipients with low immunity. Therefore, it is necessary to detect as early as possible before transplantation to provide clinicians with necessary clinical program guidance for preoperative judgment and postoperative treatment, so as to achieve the purpose of organ transplantation to cure diseases and save lives. Therefore, rapid and accurate diagnosis of pathogen infection is particularly important in the field of organ transplantation.

[0003] At present, the traditional methods of pathogen identification include: imaging, histopathology, direct culture, serological methods, drug sensitivity testing, molecular methods and NGS sequencing methods, etc. For qPCR, there is no corresponding kit on the market that can detect so many bacteria at the same time, and it is limited by the qPCR fluorescence flux and cannot detect so many pathogens at one time; for NGS testing, the testing fee is too high, about 1,000-2,000 yuan per sample, and the time required is too long, about 24-48 hours; for the gold standard culture method, the detection sensitivity is too low (about 50%), and the culture time is too long, about 2 days.

[0004] The limitations of these traditional pathogen identification methods are:

[0005] 1. These identification methods all require the cultivation of pathogenic strains, which takes a long time and is prone to contamination during the cultivation process.

[0006] 2. These identification methods can only identify a specific type of strain, and have serious lags in the diagnosis and treatment of the disease (after symptoms appear, the results cannot be known in time).

[0007] 3. Traditional bacterial molecular identification takes a long time (about 1-2 hours) to extract the bacterial genome, the steps are complicated, and improper operation during the extraction process can easily cause environmental pollution.

[0008] 4. Conventional strain identification is costly and time-consuming, and is inconvenient and uneconomical for clinical patients.

[0009] 5. For qPCR detection, due to limitations of its fluorescent channels, etc., the throughput and pathogen types that can be detected at one time are limited.

[0010] 6. It is urgent to develop a pathogen detection method that can reduce the cost of use and ensure the sensitivity and specificity of detection.

[0011] The main method of obtaining pathogen information is to use a pathogen extraction kit to extract genomic DNA from the donor's blood, tissue cells or tissue fluid and then amplify and separate the strains for testing. However, in an emergency situation where the donor organ needs to be quickly implanted into the recipient's body, this method of obtaining information on whether the pathogen is infected is limited because nucleic acid extraction and PCR amplification take about 2 hours. In addition, the method of extracting nucleic acids requires a large amount of samples, which is also a problem that needs to be solved urgently.

[0012] The existing technical solution is to first use a genome extraction kit to extract genomic DNA from the blood of the donor or recipient, and then use the genomic DNA as a template for PCR amplification, and then use sequence-specific oligonucleotide probes to specifically hybridize the PCR products, and analyze the information of the hybridization signal to obtain information on whether the sample is infected with pathogens and the type of infection. Extracting the genome from blood or human tissue takes at least 1 hour. If batch operations are performed, cross-contamination between samples due to too many samples will lead to inaccurate strain identification information. The vast majority of current PCR amplifications are amplified using Taq enzymes, which have a long amplification time (about 90 minutes) and poor fidelity. It is easy to mutate and lead to inaccurate typing; Pfu DNA polymerase (Pfu DNA polymerase), also known as Pfu polymerase, or Pfu enzyme, has both 5'-3' polymerase activity and 3'-5' exonuclease activity, and the error rate of amplified bases is 10 -6 However, the efficiency of Pfu polymerase is low. In addition, due to the poor anti-inhibition ability of the amplification buffer, the success rate of amplification of blood from different sources is low, which affects the analysis of sequencing results.

[0013] Therefore, it is necessary to develop a new rapid, high-resolution, and easy-to-operate sample strain identification and analysis method. Summary of the invention

[0014] In order to solve the problems of low throughput, long time consumption, low sensitivity, difficulty in strain identification and high cost in the prior art, the present invention provides a primer pair, a primer mixture containing the primer pair and a method for bacterial typing using the primer pair. The present invention provides a probe, a probe mixture and a method for bacterial typing using the primer pair. The probe, a probe mixture and a method for bacterial typing using the primer pair can directly use a trace amount of blood or tissue cells as a PCR amplification template, successfully amplify within 40 minutes to obtain a pathogen sequence for whether an organ is infected, and complete the infection type identification within 100 minutes.

[0015] The present invention solves the above technical problems through the following technical solutions:

[0016] A first aspect of the present invention provides a probe, wherein the nucleic acid sequence of the probe comprises a nucleic acid sequence as shown in any one of SEQ ID NOs: 5-17.

[0017] The second aspect of the present invention provides a probe composition, which comprises one or more of the probes described in the first aspect of the present invention.

[0018] In some technical schemes of the present invention, the probe composition comprises a nucleic acid sequence as shown in SEQ ID NO: 5-17.

[0019] In some technical solutions of the present invention, the 5' end of the probe is modified with C6-NH2.

[0020] The third aspect of the present invention provides a method for bacterial typing, comprising: hybridizing the PCR product of the sample to be tested with a mixture of Luminex microspheres, and then reading the fluorescence detection value using a Luminex200 instrument; wherein the Luminex microspheres are respectively labeled with the probes described in the first aspect of the present invention or the probes in the probe composition described in the second aspect of the present invention, and each probe is labeled with a microsphere of one color.

[0021] In some preferred technical solutions of the present invention, the labeling method includes forming a stable chemical bond through a dehydration condensation reaction between the carboxyl group on the Luminex microsphere and the amino group on the probe under the action of a catalyst.

[0022] In some more preferred technical embodiments of the present invention, the method further comprises PCR amplification.

[0023] In some further preferred embodiments of the present invention, the method is for non-diagnostic purposes, such as for laboratory and R&D self-testing.

[0024] In some technical schemes of the present invention, the hybridization method includes: mixing the PCR product denatured into a single strand with the mixture of the Luminex microspheres, hybridizing for 20 minutes at 55°C on a PCR instrument; adding an elution solution, centrifuging at 1300g for 5 minutes, discarding the supernatant, and repeating the elution for a total of 3 times; then adding 1x SAPE solution, labeling for 10 minutes at 55°C; adding an elution solution, centrifuging at 1300g for 5 minutes, discarding the supernatant; then adding an elution buffer and mixing.

[0025] In some technical schemes of the present invention, the sample to be tested is lymphocytes, whole blood, buccal mucosal swab, biopsy sample or frozen tissue, for example, whole blood; the bacteria are selected from one or more of Klebsiella pneumoniae, Streptococcus pneumoniae, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae and Acinetobacter baumannii.

[0026] In some technical solutions of the present invention, the PCR amplification system comprises the following components:

[0027] Primer mixture, 0.1-0.5 μM, for example 0.3 μM;

[0028] Tris-HCl, 10-30 mM, for example 20 mM;

[0029] KCl, 10-60 mM, for example 25 mM;

[0030] (NH4)2SO4, 20-60 mM, for example 20 mM;

[0031] MgSO4, 0.5-5 mM, for example 2.5 mM;

[0032] MgCl2, 0.5-3 mM, for example 1.5 mM;

[0033] dNTP, 0.1-0.5 mM, for example 0.25 mM;

[0034] dUTP, 0.3-0.8 mM, for example 0.5 mM;

[0035] Betaine, 10-25 mM, for example 12.5 mM;

[0036] Trehalose, 5-25 mM, for example 20 mM;

[0037] Glycerol, 1-5%, e.g. 2.5%;

[0038] BSA, 0.5%-5%, such as 0.5%, 1%, 2%, 3%, 4% or 5%;

[0039] Surfactant, 0.05%-0.5%, such as 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%;

[0040] DNA polymerase, 1U-3U per 20μL system, for example 2U;

[0041] UDG enzyme, 0.5-3U per 50μL system, for example 1U;

[0042] The PCR amplification procedure includes one or more of the following:

[0043] 20-30°C, 2-10 min, e.g. 25°C, 3 min;

[0044] 92-98°C, 2-10 min, e.g. 95°C, 2 min;

[0045] 92-98°C, 3-15s, e.g. 95°C, 5s;

[0046] 50-60℃, 5-30s, e.g. 58℃, 5s;

[0047] 72°C, 5-30s, e.g. 72°C, 10s;

[0048] The last three steps are repeated 30-45 times, for example 35 times.

[0049] In some technical schemes of the present invention, the primer mixture comprises one or more of the nucleic acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4; the surfactant is Triton-X100; the DNA polymerase comprises one or more of hanta, Primerstar, Klenow, Bst, Phi29, KOD enzyme, Taq DNA enzyme and Pfu polymerase, for example, Taq DNA enzyme and Pfu polymerase.

[0050] In some preferred technical solutions of the present invention, the 5' end of the primer in the primer mixture is modified with biotin; and the enzyme activity ratio of the Taq DNA enzyme to the Pfu polymerase is 2:8-9:1.

[0051] In some more preferred technical solutions of the present invention, the enzyme activity ratio of the Taq DNA enzyme to the Pfu polymerase is 7:3 to 9:1, for example, 7:3, 8:2 or 9:1.

[0052] The fourth aspect of the present invention provides a kit comprising one or more of the probes described in the first aspect of the present invention, the probe mixture described in the second aspect of the present invention, and the mixture of Luminex microspheres in the method described in the third aspect of the present invention.

[0053] In some technical schemes of the present invention, the kit further comprises one or more of the following: MES, 1×TE at pH 8.0, streptavidin-phycoerythrin, hybridization solution, etc., such as the primer mixture, positive control and DNA positive template in the method described in the third aspect of the present invention.

[0054] In some technical solutions of the present invention, the kit further comprises the PCR amplification system as described in the method according to the third aspect of the present invention.

[0055] The fifth aspect of the present invention provides the use of one or more of the probe according to the first aspect of the present invention, the probe mixture according to the second aspect of the present invention, and the kit according to the fourth aspect of the present invention in bacterial typing.

[0056] In some preferred technical solutions of the present invention, the bacteria are selected from one or more of Klebsiella pneumoniae, Streptococcus pneumoniae, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae and Acinetobacter baumannii.

[0057] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0058] The reagents and raw materials used in the present invention are commercially available.

[0059] The positive and progressive effects of the present invention are as follows: the probes and probe mixtures provided by the present invention can significantly distinguish between positive and negative samples, thereby improving the specificity of detection; the bacterial typing method provided by the present invention can directly use a trace amount of blood or tissue cells as a PCR amplification template, successfully amplify within 40 minutes to obtain the pathogen sequence for whether the organ is infected, and complete the infection type identification within 100 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The figure is the electrophoresis diagram of the amplified product. Marker: 50, 100, 200, 300, 400, 500 bp.

[0061] Figure 2 The figure is the electrophoresis diagram of the amplified product. Marker: 50, 100, 200, 300, 400, 500 bp.

[0062] Figure 3 The electrophoresis diagram of the amplified product. DETAILED DESCRIPTION

[0063] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are usually performed under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0064] As used herein, a "biological sample" can be, for example, lymphocytes, whole blood, buccal mucosal swabs, biopsy samples, perfusate samples, drainage samples, or frozen tissue or any other sample containing genomic DNA. Although nearly any tissue source can be used for molecular genotyping, lymphocytes, perfusate samples, drainage samples, for example, from peripheral blood are most commonly used. Samples obtained by non-invasive methods, such as by cheek swab or saliva-based DNA collection, can also be used.

[0065] "DNA" refers to various forms of deoxyribonucleic acid known in the art, such as genomic DNA. A variety of suitable methods for extracting DNA from these sources are known in the art. These methods range from organic solvent extraction to adsorption on silica-coated magnetic beads, cation exchange columns, and enzymatic extraction of DNA. Automated DNA extraction systems are also commercially available and can provide high-quality, high-purity DNA.

[0066] As used herein, "polymerase chain reaction" or PCR is the amplification of nucleic acids, consisting of an initial denaturation step that separates the strands of a double-stranded nucleic acid sample, followed by repeated (i) annealing steps that allow the amplification primers to anneal specifically to the flanking positions of the target sequence; (ii) an extension step that extends the primers in the 5' to 3' direction thereby forming an amplicon polynucleotide complementary to the target sequence, and (iii) a denaturation step that causes the amplicon to separate from the target sequence (Mullis et al., eds., The Polymerase Chain Reaction, Birk Hauser, Boston, Mass. (1994)). Each of the above steps can be performed at different temperatures, preferably using an automated thermal cycler (Applied Biosystems LLC, a division of Life Technologies Corporation, Foster City, CA.). If desired, the RNA sample can be converted to a DNA / RNA heteroduplex or double-stranded cDNA by methods known to those skilled in the art. PCR methods also include reverse transcriptase-PCR and other reactions that follow the principles of PCR.

[0067] As used herein, "amplification" refers to a wide range of techniques that increase polynucleotide sequences linearly or exponentially. Exemplary amplification techniques include, but are not limited to, PCR or any other method using a primer extension step. Other non-limiting examples of amplification include, but are not limited to, ligase detection reaction (LDR) and ligase chain reaction (LCR). Amplification methods can include thermal cycling or can be performed isothermally. In various specific embodiments, the term "amplification product" includes products from any amplification reaction cycle number.

[0068] In certain embodiments, the amplification method includes at least one amplification cycle, such as but not limited to the following continuous process: hybridizing a primer to a primer-specific portion of a target sequence or amplifying a product from any number of amplification reaction cycles; using a polymerase to synthesize a chain of nucleotides in a template-dependent manner; and denaturing the newly formed nucleic acid duplex to separate the chains. The cycle may or may not be repeated.

[0069] There are many known methods for amplifying nucleic acid sequences, including, for example, PCR. For example, PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (ed. Innis et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991)); PCR (ed. McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,1594,965,188 and 5,333,675, each of which is incorporated herein by reference in its entirety for all purposes.

[0070] Nucleic acid amplification techniques are traditionally classified according to the temperature requirements of the amplification method. Isothermal amplification is performed at a constant temperature, in contrast to amplification that requires cycling between high and low temperatures. Examples of isothermal amplification techniques are: Strand Displacement Amplification (SDA; Walker et al., 1992, Proc. Natl. Acad. Sci. USA 89:392-396; Walker et al., 1992, Nuc. Acids. Res. 20:1691-1696; and EP 0 497 272), self-sustained sequence replication (3SR; Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), the Q.beta. replicase system (Lizardi et al., 1988, BioTechnology 6:1197-1202), and the techniques disclosed in WO 90 / 10064 and WO 91 / 03573.

[0071] "Preferred" and "preferably" refer to specific embodiments of the present invention that may provide certain benefits in certain circumstances. However, other specific embodiments may also be preferred under the same or other circumstances. In addition, the detailed description of one or more preferred specific embodiments does not mean that other specific embodiments are useless and is not used to exclude other specific embodiments from the scope of the present invention.

[0072] The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0073] Also herein, numerical ranges recited by endpoints include all values ​​subsumed within that range.The term "and / or" means one or all of the listed elements or any combination of two or more of the listed elements.

[0074] Example 1 Effects of different volume ratios of Triton X-100 and different concentrations of BSA on PCR amplification

[0075] 1.1 Experimental materials are shown in Table 1.

[0076] Table 1 Experimental materials

[0077]

[0078]

[0079] The primer sequences corresponding to bacterial amplification are shown in Table 2.

[0080] Table 2 Primer sequences

[0081]

[0082] 1.2 Experimental section

[0083] a) First, prepare a 2× concentrated solution according to the concentrations of the following components: 40mM Tris-HCl, 50mM KCl, 40mM (NH4)2SO4, 5mM MgSO4, 3mM MgCl2, 0.5mM dNTP, 1mM dUTP, 25mM betaine, 40mM trehalose, 5% glycerol, final reaction concentration of UDG enzyme 1U / 50μL, pH 8.3, then add different concentrations of BSA and Triton X-100 to the above buffer, and the working concentration combination is shown in Table 3.

[0084] Table 3 Working concentration combinations

[0085] Combination No. 1 2 3 4 5 6 BSA concentration 0.5％ 1％ 2％ 3％ 4％ 5％ Triton-X 100 (v / v) 0.05％ 0.1％ 0.2％ 0.3％ 0.4％ 0.5％

[0086] b) Prepare the PCR amplification system as shown in Table 4.

[0087] Table 4 PCR amplification system

[0088] Components Volume (μL) concentration Buffer 12.5 1× Enzyme mixture 0.4 2U Primer mix 2 0.3μM Different combinations of BSA and Triton X-100 2 Ultrapure water 6.1 Whole blood 2

[0089] c) PCR amplification

[0090] The amplification program is shown in Table 5.

[0091] Table 5 Amplification procedures

[0092]

[0093] The amplified product was analyzed by electrophoresis. Figure 1 As shown in the figure, from the electrophoresis amplification test results, it can be seen that combination 2, combination 3, and combination 4 have better amplification results. This may be because the concentration of the surfactant in combination 1 is too low, which is not enough to lyse the cells and release DNA. The surfactant concentration in combination 5 and combination 6 is too high, which leads to reduced activity of DNA polymerase and relatively reduced amplification efficiency.

[0094] Example 2 Adjusting the amount of two DNA polymerases to fully shorten the PCR amplification time

[0095] 2.1 Experimental materials see Example 1.

[0096] 2.2 Experimental part

[0097] a) First, prepare a 2× concentrate according to the concentrations of the following components: 40 mM Tris-HCl, 50 mM KCl, 40 mM (NH4)2SO4, 5 mM MgSO4, 3 mM MgCl2, 0.5 mM dNTP, 1 mM dUTP, 0.4% (v / v) Triton-X 100 surfactant, 4% BSA, 25 mM betaine, 40 mM trehalose, 5% glycerol, and the final reaction concentration of UDG enzyme was 1 U / 50 μL, pH 8.3, and then mix Taq enzyme and Pfu enzyme according to Table 6.

[0098] Table 6 Amounts of Taq enzyme and Pfu enzyme

[0099] Combination No. 1 2 3 4 5 6 7 8 Taq(μL) 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Pfu(μL) 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 Total volume 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00

[0100] b) Prepare the PCR amplification system as shown in Table 7.

[0101] Table 7 PCR amplification system

[0102] Components Volume (μL) concentration Buffer 12.5 1× Enzyme mixture 0.4 2U Primer mix 2 0.3μM Ultrapure water 8.1 Whole blood 2

[0103] c) PCR amplification

[0104] The amplification program is shown in Table 5.

[0105] The amplified product was analyzed by electrophoresis. Figure 2 From the electrophoresis amplification test results, it can be seen that combination 6, combination 7, and combination 8 have better amplification results, that is, when the ratio of Taq enzyme to Pfu enzyme is between 7:3 and 9:1, they can be amplified well under the rapid PCR program.

[0106] Example 3 Rapid blood direct amplification followed by detection using Luminex instrument

[0107] 3.1 Experimental section

[0108] a) Preparation of PCR blood direct amplification buffer: First, prepare a 2× concentrate according to the concentration of each component below. The working concentration of each component is:

[0109] 20mM Tris-HCl, 25mM KCl, 20mM (NH4)2SO4, 2.5mM MgSO4, 1.5mM MgCl2, 0.25mMdNTP, 0.5mM dUTP, 0.2% (v / v) Triton-X 100 surfactant, 2% BSA, 12.5mM betaine, 20mM trehalose, 2.5% glycerol, final reaction concentration UDG enzyme 1U / 50μL, pH 8.3.

[0110] Therefore, the preparation concentration of 2× concentrated solution is: 40mM Tris-HCl, 50mM KCl, 40mM (NH4)2SO4, 5mMMgSO4, 3mM MgCl2, 0.5mM dNTP, 1mM dUTP, 0.4% (v / v) Triton-X 100 surfactant, 4% BSA, 25mM betaine, 40mM trehalose, 5% glycerol, final reaction concentration UDG enzyme 1U / 50μL, pH 8.3.

[0111] b) Mixing of Pfu enzyme and Taq enzyme

[0112] The two DNA polymerases were mixed in a ratio of 2:8 between Pfu enzyme and Taq enzyme activity, and the mixture was centrifuged and set aside. Since both enzymes were 5U / μL, they only needed to be mixed in a volume ratio of 2:8.

[0113] c) Prepare the PCR amplification system as shown in Table 8.

[0114] Table 8 PCR amplification system

[0115] Components Volume (μL) concentration PCR Blood Direct Amplification Buffer 12.5 1× Enzyme mixture 0.4 2U Primer mix 2 0.3μM Ultrapure water 8.1 Whole blood 2

[0116] d) PCR amplification

[0117] The amplification program is shown in Table 5.

[0118] The amplified products were analyzed by electrophoresis. Figure 3 As shown, labels 1-5 represent direct amplification from blood, and M represents protein marker.

[0119] The information of DNA probes labeled with Luminex microspheres is shown in Table 9.

[0120] Table 9 Luminex microsphere labeled DNA probe information

[0121]

[0122]

[0123] For Luminex microsphere labeled probe method, refer to Luminex microsphere instruction manual - xMAP Cookbook 4th Edition Chapter 5.2.

[0124] The result interpretation chart displayed by the corresponding probes is shown in Table 10.

[0125] Table 10 The corresponding probes show the result interpretation spectrum

[0126]

[0127] Test result determination:

[0128] 1. Each microbead of each sample contains FI (fluorescence intensity), and the positive percentage value is calculated according to the following formula:

[0129] Positive percentage value = 100 × [FI (each probe) - FI (probe negative control) / FI (probe positive control) - FI (probe negative control)]

[0130] A probe's positive percentage value higher than the probe's "cut-off" value indicates a positive reaction. A probe's positive percentage value lower than the probe's "cut-off" value indicates a negative reaction.

[0131] 2. For each sample test result, if the FI value of the positive probe is less than 1000, the sample is judged to be negative in this experiment, the result is not considered accurate, and the experiment needs to be repeated.

[0132] For samples with positive probe FI values ​​greater than 1000, they are considered normal samples, and the positive percentage value of each probe can be calculated;

[0133] 3. Compare the positive percentage value and "cut-off" value of each microsphere. The positive percentage value of the probe is higher than the "cut-off" value of the probe, which is a positive reaction. The positive percentage value of the probe is lower than the "cut-off" value of the probe, which is a negative reaction. The FI of the positive control probe should be above 1000. Each probe is expressed as a percentage of the positive control. The "cut-off" value of each probe is obtained in advance by performing more than 100 sample DNA analysis experiments.

[0134] e) After PCR amplification, the PCR product was centrifuged instantaneously, and 5 μL of the PCR product supernatant was taken for denaturation and neutralization, and typing was performed using a Luminex200 instrument. The typing information and reference information are shown in Table 11.

[0135] Table 11 Typing information and reference information

[0136] Sample No. Analytical Information Reference Information (NGS Test Results) 1 Klebsiella pneumoniae Klebsiella pneumoniae 2 Staphylococcus aureus Staphylococcus aureus 3 Acinetobacter baumannii Acinetobacter baumannii 4 Pseudomonas aeruginosa Pseudomonas aeruginosa 5 Untargeted detection of pathogens Candida albicans

[0137] The results showed that the typing results were consistent with the reference information, and successfully achieved rapid identification of whether the blood sample contained strains and the type of strain sample within 2 hours using blood as a template.

[0138] Example 4 Single Probe Optimization

[0139] For the experimental method, see Example 3. 5×10 7 The number of copies / mL was gradually diluted to 5×10 3copies / mL as the positive sample template. The negative sample is a blood sample collected from a healthy volunteer. The above-mentioned diluted template and negative sample were amplified using the above method. The probes were optimized and compared one by one, that is, the three probes PR1, PR1-1, and PR1-2 were labeled at the same time, and the comparison results of the same negative and positive samples were obtained by detecting them at the same time; PR2, PR3, etc. were detected in turn. For PR11 and PR12 as positive internal reference probes, the designed signal value meets the signal value requirements for detection, and the signal value is high, so no optimization is performed. For PR13 as a negative internal reference probe, the design meets the requirements for detection, so no optimization is performed. The DNA probe information labeled with Luminex microspheres and the signal value results of the microspheres are shown in Table 12.

[0140] As shown in Table 12, the probe comparison of PR1-PR3 and PR7-PR10 shows that the probe sequences with significantly improved positive signal values ​​and more significant positive and negative signal value distinction are selected as the optimized combination probes. The comparison data of the three probe groups PR4, PR5, and PR6 before and after optimization show that the effect difference is not as significant as that of other groups, but the positive signal values ​​have all been improved.

[0141] Table 12 Luminex microsphere labeled DNA probe information

[0142]

[0143]

[0144] Example 5 Probe combination optimization

[0145] For the experimental method, see Example 3. 5×10 7 The number of copies / mL was gradually diluted to 5×10 6 copies / mL, 5×10 5 copies / mL, 5×10 4 copies / mL, 5×10 3 copies / mL, 1×10 3 copies / mL, 5×10 2copies / mL as a template. Negative samples are blood samples collected from healthy volunteers. Partially inactivated common infectious pathogens, including Mycobacterium tuberculosis, Chlamydia pneumoniae, Candida albicans, Aspergillus fumigatus, and Mycoplasma pneumoniae, were selected for specific detection. The above-mentioned diluted templates, negative samples, and specific detection samples were amplified respectively using the above method. The DNA probe combination information labeled with Luminex microspheres and the signal value results of the microspheres are shown in Table 13. The results are shown in the table below. For the primer probe combination shown in combination 1, the accuracy, sensitivity, and specificity of the detected samples are better. Therefore, the probe combination of combination 1 was selected as the subsequent test.

[0146] Table 13 Probe combination optimization

[0147]

[0148] Example 6: Sensitivity Detection

[0149] 5×10 of the self-made pathogen target (including various pathogen subtypes) 7 The number of copies / mL was gradually diluted to 5×10 6 copies / mL, 5×10 5 copies / mL, 5×10 4 copies / mL, 5×10 3 copies / mL, 1×10 3 copies / mL, 5×10 2 copies / mL as template. Negative samples are blood samples collected from healthy volunteers. The diluted template and negative sample are amplified using the above method, and the result judgment criteria are consistent with the above judgment method. The probe combination used is PR1-PR13. As can be seen from the table below, in the case of low concentration template, this method can still be detected, so it has a high sensitivity. At the same time, when using the method of the present invention, there is no cross reaction between the probes, with high sensitivity and consistency. The results are shown in Table 14.

[0150] Table 14 Sensitivity test results

[0151]

[0152] Example 7 For specific detection

[0153] Partially inactivated common infectious pathogens were selected, including Mycobacterium tuberculosis, Chlamydia pneumoniae, Candida albicans, Aspergillus fumigatus, and Mycoplasma pneumoniae. The probe combination used was PR1-PR13, and specific detection was performed. The detection process and result determination were as described above, proving that the present invention had no cross-reaction with other pathogenic nucleic acids in the examples. The results are shown in Table 15.

[0154] Table 15 Specificity test results

[0155] sample concentration result Remark Mycobacterium tuberculosis <![CDATA[4.32×10 4 copies / μL]]> No target strains were detected Inactivated Standards Chlamydia pneumoniae <![CDATA[3.34×10 4 copies / μL]]> No target strains were detected Inactivated Standards Candida albicans <![CDATA[8.96×10 4 copies / μL]]> No target strains were detected Inactivated Standards Aspergillus fumigatus <![CDATA[7.64×10 4 copies / μL]]> No target strains were detected Inactivated Standards Mycoplasma pneumoniae <![CDATA[2.06×10 4 copies / μL]]> No target strains were detected Inactivated Standards

[0156] Conclusion: The detection kit provided by the present invention can detect multiple pathogens simultaneously and rapidly, and can stably detect low concentrations of pathogens. This method can be adjusted accordingly according to actual clinical needs (for example, increasing the probe sequence, correspondingly increasing the detection objects, etc.). This method uses two labeled microsphere probes to detect a pathogen, which greatly improves the accuracy of detection, avoids false negative and false positive results caused by randomness and sample interference, and has a wide range of uses and broad application prospects.

Claims

1. A probe, characterized in that: The nucleic acid sequence of the probe comprises a nucleic acid sequence as shown in any one of SEQ ID NOs: 5-17.

2. A probe composition, characterized in that: The probe composition comprises one or more of the probes described in claim 1; Preferably, the probe composition comprises the nucleic acid sequence shown in SEQ ID NO: 5-17; More preferably, the 5' end of the probe is modified with C6-NH2.

3. A method for bacterial typing, characterized in that: The method comprises: after hybridizing the PCR product of the sample to be tested with a mixture of Luminex microspheres, using a Luminex200 instrument to read the fluorescence detection value; wherein the Luminex microspheres are respectively labeled with the probes according to claim 1 or the probes in the probe composition according to claim 2, and each probe is labeled with a microsphere of one color; Preferably, the labeling method includes forming a stable chemical bond by a dehydration condensation reaction between the carboxyl group on the Luminex microsphere and the amino group on the probe under the action of a catalyst; More preferably, the method further comprises PCR amplification; Even more preferably, the method is for non-diagnostic purposes.

4. The method according to claim 3, characterized in that The hybridization method comprises: mixing the PCR product denatured into a single strand with the mixture of the Luminex microspheres, performing a hybridization reaction at 55° C. for 20 minutes on a PCR instrument; adding an elution solution, centrifuging at 1300 g for 5 minutes, discarding the supernatant, and repeating the elution for a total of 3 times; then adding a 1x SAPE solution, performing a labeling reaction at 55° C. for 10 minutes; adding an elution solution, centrifuging at 1300 g for 5 minutes, discarding the supernatant; and then adding an elution buffer and mixing.

5. The method according to claim 4, characterized in that The sample to be tested is lymphocyte, whole blood, buccal mucosal swab, biopsy sample or frozen tissue; the bacteria are selected from one or more of Klebsiella pneumoniae, Streptococcus pneumoniae, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae and Acinetobacter baumannii.

6. The method according to any one of claims 3 to 5, characterized in that: The PCR amplification system comprises the following components: primer mix, 0.1–0.5 μM; Tris-HCl, 10-30 mM; KCl, 10-60 mM; (NH4)2SO4, 20-60mM; MgSO4, 0.5-5 mM; MgCl2, 0.5-3 mM; dNTP, 0.1-0.5 mM; dUTP, 0.3-0.8 mM; Betaine, 10-25 mM; Trehalose, 5-25 mM; Glycerol, 1-5%; BSA, 0.5%-5%; Surfactant, 0.05%-0.5%; DNA polymerase, 1U-3U per 20μL system; UDG enzyme, 0.5-3U per 50μL system; The PCR amplification procedure includes one or more of the following: 20-30℃, 2-10min; 92-98℃, 2-10min; 92-98℃，3-15s； 50-60℃，5-30s； 72℃，5-30s； Among them, the last three steps are cycled 30-45 times.

7. The method according to claim 6, characterized in that The primer mixture comprises one or more of the nucleic acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; the surfactant is Triton-X 100; the DNA polymerase comprises one or more of hanta, Primerstar, Klenow, Bst, Phi29, KOD enzyme, Taq DNA enzyme and Pfu polymerase; Preferably, the 5' end of the primer in the primer mixture is modified with biotin; the enzyme activity ratio of the Taq DNA enzyme to the Pfu polymerase is 2:8 to 9:1; More preferably, the enzyme activity ratio of the Taq DNA enzyme to the Pfu polymerase is 7:3 to 9:

1.

8. A kit, characterized in that: The kit comprises one or more of the probe according to claim 1, the probe mixture according to claim 2, and the mixture of Luminex microspheres in the method according to claim 3; Preferably, the kit further comprises one or more of the following: MES, 1×TE at pH 8.0, streptavidin-phycoerythrin, hybridization solution, etc., the primer mixture, positive control and DNA positive template in the method of claim 7.

9. The kit according to claim 8, characterized in that The kit further comprises the PCR amplification system according to the method of claim 6 or 7.

10. Use of one or more of the probe according to claim 1, the probe mixture according to claim 2, and the kit according to claim 8 or 9 in bacterial typing; Preferably, the bacteria are selected from one or more of Klebsiella pneumoniae, Streptococcus pneumoniae, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae and Acinetobacter baumannii.

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