A primer probe combination, method and application for detecting pathogenic microorganisms

Abstract

This invention discloses a primer-probe combination, method, and application for detecting pathogenic microorganisms. The nucleic acid sequences of the primers include those shown in SEQ ID NO.1-SEQ ID NO.42; the nucleic acid sequences of the fluorescent probes include those shown in SEQ ID NO.43-SEQ ID NO.63. This invention, through a primer design approach combining conserved and specific primers, and based on one-step quantitative real-time PCR technology, achieves rapid differentiation of pathogen types in patients. It has a wide target coverage, high detection efficiency, and ensures stable binding of the probe to the target fragment during primer extension, resulting in high accuracy and effectively avoiding cross-contamination and aerosol contamination.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and molecular biology, and relates to a primer-probe combination, method and application for detecting pathogenic microorganisms. Background Technology

[0002] Infectious diseases are mainly classified into bacterial infections, fungal infections, and viral infections (DNA viruses and RNA viruses). Pathogen detection is a crucial step in the diagnosis and treatment of infectious diseases. Different types of pathogen infections require significantly different treatment approaches in clinical practice. For example, bacterial pneumonia requires antibiotic treatment, fungal pneumonia commonly uses antifungal drugs, and viral pneumonia emphasizes supporting the patient's own immunity. Therefore, early pathogen detection can assist in developing a treatment plan and improve patient survival rates.

[0003] Currently, commonly used clinical methods for detecting infectious pathogens include complete blood count (CBC), serological diagnosis, pathogen isolation and culture, and nucleic acid testing. CBC assesses blood quality and identifies diseases by statistically analyzing changes in the number and morphological distribution of blood cells. Based on the results, it can preliminarily diagnose whether a patient has a bacterial or viral infection. However, the accuracy of CBC is affected by various physiological factors and cannot differentiate between fungal infections, limiting its clinical application. Serological diagnosis involves detecting corresponding antibodies in a patient's serum using known antigens. While this method is highly accurate, its long clinical turnaround time affects patient treatment efficiency. Pathogen isolation and culture involves culturing pathogens in vitro and then performing biological identification. This method is low-cost but time-consuming and prone to bias. Nucleic acid testing detects infectious pathogens at the nucleic acid level, including singleton or multiplex quantitative PCR and sequencing. Singleton or multiplex PCR can accurately detect pathogen species, but their coverage is limited, requiring physicians to pre-determine the patient's likely infection direction before deciding on the PCR target range. Currently, the most commonly used sequencing methods in clinical practice are first-generation sequencing (1G) and second-generation high-throughput sequencing (NGS). 1G sequencing typically sequences the PCR amplicon of pathogenic microorganisms and can be further divided into tNGS and mNGS. tNGS is consistent with multiplex PCR and requires prior determination of the infection extent. Although mNGS can perform full sequencing of the pathogen infecting a patient, it is more expensive and has a longer clinical turnaround time, making it unsuitable for the preliminary diagnosis of pathogen type.

[0004] In summary, current methods for detecting pathogenic microorganisms suffer from limitations such as limited coverage, the need for pre-determining the extent of infection, high costs, long clinical turnaround times, and unsuitability for preliminary diagnosis of pathogen types. Developing a pathogenic microorganism detection method that offers broad coverage, high accuracy, and high sensitivity has become one of the most pressing issues to be addressed in the fields of biotechnology and molecular biology. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a primer-probe combination, method, and application for detecting pathogenic microorganisms. It develops a one-step multiplex quantitative PCR detection method for pathogenic microorganisms (bacteria, fungi, DNA viruses, and RNA viruses) with broad coverage, short processing time, and high accuracy. This method alleviates the pressure on current clinical pathogen detection and diagnosis and can assist doctors in formulating preliminary treatment plans for infectious patients before admission.

[0006] To achieve this objective, the present invention employs the following technical solution:

[0007] In a first aspect, the present invention provides primers and fluorescent probes for detecting pathogenic microorganisms, wherein the nucleic acid sequences of the primers include the sequences shown in SEQ ID NO.1-SEQ ID NO.42; and the nucleic acid sequences of the fluorescent probes include the sequences shown in SEQ ID NO.43-SEQ ID NO.63.

[0008] This invention utilizes a primer design approach that combines conserved and specific methods, based on one-step quantitative real-time PCR technology, to achieve rapid differentiation of pathogen types in patients. It has a wide target range, high detection efficiency, and the probe maintains stable binding with the target fragment during primer extension, resulting in high accuracy and effectively avoiding cross-contamination and aerosol contamination.

[0009] The primers and probes designed in this invention are shown in Table 1.

[0010] Table 1

[0011]

[0012]

[0013]

[0014]

[0015] The one-step multiplex quantitative PCR method used in this invention is a multi-target quantitative detection method based on multiplex PCR and one-step quantitative PCR. It combines the high-efficiency quantitative capability of one-step quantitative PCR with the multi-target detection capability of multiplex PCR, enabling the amplification and fluorescence detection of multiple target genes or fragments in a single reaction tube. However, achieving this high-throughput detection depends on the precise design of different target primers and probes to prevent non-specific amplification and primer dimers. In addition, the annealing temperature also needs to be strictly controlled.

[0016] Preferably, the fluorescent probe has a fluorescent group at its 5' end and a quenching group at its 3' end.

[0017] Preferably, the fluorescent group includes any one or a combination of at least two of FAM, Cy5, VIC or ROX.

[0018] Preferably, the quenching group includes any one or a combination of at least two of BHQ1, BHQ2, or MGB.

[0019] Preferably, the pathogenic microorganism includes any one or a combination of at least two of bacteria, fungi, DNA viruses, or RNA viruses.

[0020] Preferably, the fungus includes any one or a combination of at least two of the genera Cryptococcus, Candida, Aspergillus, or Pneumocystis.

[0021] Preferably, the DNA virus includes any one or a combination of at least two of the following: human herpesvirus I, human herpesvirus II, human herpesvirus III, human herpesvirus IV, human herpesvirus V, hepatitis B virus, human parvovirus B19, or all types of adenovirus.

[0022] Preferably, the RNA virus includes any one or a combination of at least two of the following: rhinovirus, influenza A virus, influenza B virus, coronavirus 229E, coronavirus OC43, coronavirus HKU1, coronavirus NL63, novel coronavirus, or enterovirus.

[0023] Secondly, the present invention provides the application of the primers and fluorescent probes described in the first aspect for detecting pathogenic microorganisms in the preparation of products for detecting pathogenic microorganisms.

[0024] Thirdly, the present invention provides a kit for detecting pathogenic microorganisms, the kit comprising the primers and fluorescent probes for detecting pathogenic microorganisms described in the first aspect.

[0025] Fourthly, the present invention provides the application of the primers and fluorescent probes described in the first aspect for detecting pathogenic microorganisms in the detection of pathogenic microorganisms.

[0026] Fifthly, the present invention provides a method for detecting pathogenic microorganisms, the method comprising the following steps:

[0027] (1) Extract DNA from the sample to be tested as a template, and perform one-step real-time PCR amplification using the primers and fluorescent probes for detecting pathogenic microorganisms as described in claim 1 or 2;

[0028] (2) Collect and detect the fluorescence signal of each channel in each reaction well during PCR amplification to generate an amplification curve;

[0029] (3) Determine whether the sample contains pathogenic microorganisms based on the Ct value of the fluorescence signal in each channel.

[0030] Preferably, the pathogenic microorganism includes any one or a combination of at least two of bacteria, fungi, DNA viruses, or RNA viruses.

[0031] Preferably, the fungus includes any one or a combination of at least two of the genera Cryptococcus, Candida, Aspergillus, or Pneumocystis.

[0032] Preferably, the DNA virus includes any one or a combination of at least two of the following: human herpesvirus I, human herpesvirus II, human herpesvirus III, human herpesvirus IV, human herpesvirus V, hepatitis B virus, human parvovirus B19, or all types of adenovirus.

[0033] Preferably, the RNA virus includes any one or a combination of at least two of the following: rhinovirus, influenza A virus, influenza B virus, coronavirus 229E, coronavirus OC43, coronavirus HKU1, coronavirus NL63, novel coronavirus, or enterovirus.

[0034] Preferably, each reaction well in step (2) contains at least four fluorescent channels, each fluorescent channel corresponding to a type of pathogen.

[0035] Preferably, each reaction well in step (2) further includes four fluorescence channels: FAM, ROX, VIC, and Cy5.

[0036] Preferably, the criterion for judgment in step (3) is:

[0037] A Ct value <28 for the fluorescent channel used to detect bacteria indicates a positive result for bacteria.

[0038] A fluorescence channel Ct value <33 for fungal detection is considered positive for fungi.

[0039] A Ct value <30 for the fluorescent channel used to detect DNA viruses indicates a positive result for DNA viruses.

[0040] A fluorescence channel Ct value <30 for detecting RNA viruses indicates a positive result for RNA viruses.

[0041] Preferably, the sample to be tested includes any one of the following: blood, bronchoalveolar lavage fluid, nasopharyngeal swab, sputum, or cerebrospinal fluid.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) This invention utilizes a primer design approach that combines conserved and specific primers, based on one-step real-time quantitative PCR technology, to achieve rapid differentiation of pathogen types in patients. The target range is broad, encompassing almost all clinically common bacteria and fungi, as well as more than thirty common DNA or RNA viruses. A single real-time quantitative PCR test can simultaneously differentiate four pathogen types, revealing the specific infectious disease and the presence of mixed infections, significantly reducing detection time and improving detection efficiency.

[0044] (2) All primers of this invention have similar Tm values, which are all between 56-60℃, and all probes have Tm values ​​between 65-70℃, which are 7-10℃ higher than the primers, which can ensure that the probes maintain stable binding with the target fragment during primer extension;

[0045] (3) The PCR program includes the amplification system and the reverse transcription process, enabling the entire process to be carried out in a single tube. There is no need to perform reverse transcription of RNA separately, and no additional tube opening or pipetting operations are required, which simplifies the operation process and can effectively avoid cross-contamination and aerosol contamination. Attached Figure Description

[0046] Figure 1 This is a peak shape detection diagram of the amplification products of the primers designed in this invention;

[0047] Figure 2 This is a peak shape detection diagram of the amplification products from primers with poor performance;

[0048] Figure 3 This is a graph showing the RPM distribution of target sequences after high-throughput sequencing of commercial plasmids. Detailed Implementation

[0049] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0050] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0051] Example 1

[0052] Validation of primer amplification effect.

[0053] The amplification capabilities of 21 primer pairs, from SEQ ID NO.1 to SEQ ID NO.63, were validated (the experiments included backup primers designed in the early stages).

[0054] A commercially available plasmid containing the target sequence (PUC57 Shanghai Sangon Biotech, contract number: GN202318177) was used as a template to amplify and detect each target primer.

[0055] The primers used in the examples include all primer sequences in Table 1 and primer sequences with poor performance that are not included in Table 1.

[0056] The amplification reaction solution used in Example 1 was a commercially available general-purpose premixed reaction solution, which should include Taq enzyme, KCl, MgCl2, and dNTPs. The dNTPs include dATP, dGTP, dCTP, and dTTP.

[0057] The specific steps of Example 1 are as follows:

[0058] (1) Dilute each primer to a working concentration of 10 μmol / L according to the instructions of the synthesis supplier.

[0059] (2) The concentration of the commercial plasmid was determined using a spectrophotometer and the commercial plasmid was diluted to 0.2 ng / μL.

[0060] (3) Prepare the PCR reaction system according to Table 2 below. The amplification premixed reaction solution is a commercially available conventional PCR amplification reaction solution. The one used in this invention is KAPA HIFI Hot Start PCR Mix (Roche). Any commercially available premixed reaction solution with polymerase chain reaction capability can be substituted.

[0061] Table 2

[0062] Components Volume (μL) Amplification premixed reaction solution (2×mix) 12.5 Target primer F 0.75 Target primer R 0.75 Template (commercial plasmid nucleic acid) 2 Nuclease-free water Add to 25

[0063] (4) Set the PCR instrument program according to Table 3 below to perform the amplification reaction.

[0064] Table 3

[0065]

[0066]

[0067] (5) The experimental target and theoretical fragment information are shown in Table 4. The amplified fragments were detected using the Qsep-100 fully automated nucleic acid analyzer.

[0068] Table 4

[0069]

[0070]

[0071] (6) Fragment detection results are as follows Figure 1As shown, the amplification products of Q2-Q22 all have strong specificity, with sharp peaks and no other impurities.

[0072] (7) As shown in Table 5 below, sequences with low GC content and potential dimer structures with other primers were detected, and the results are as follows. Figure 2 As shown, some peaks failed to amplify, while others exhibited impurities or dimers. Subsequent one-step multiplex qPCR amplification was performed on these poorly performing target-probe combinations, with the results as follows: Figure 3 As shown, the primer-probe combination for these targets is ineffective, and the sequences in Table 5 are replaced with the corresponding sequences in Table 1.

[0073] Table 5

[0074]

[0075] Example 2

[0076] One-step multiplex quantitative PCR target amplification detection.

[0077] Using a commercial plasmid (PUC57 Shanghai Sangon Biotech, contract number: GN202318177) as a template, a one-step multiplex real-time PCR detection was performed using the primers and probes designed in this invention.

[0078] The one-step multiplex fluorescence quantitative reaction solution used in Example 2 is a commercially available general-purpose premixed reaction solution.

[0079] The specific steps of Example 2 are as follows:

[0080] (1) Dilute each primer to a working concentration of 10 μmol / L according to the instructions of the synthesis supplier.

[0081] (2) Dilute each probe to a working concentration of 10 μmol / L according to the instructions of the synthesis supplier.

[0082] (3) The concentration of the commercial plasmid was determined using a spectrophotometer and diluted to 0.2 ng / μL.

[0083] (4) Configure the one-step multiplex real-time PCR reaction system according to Table 6 below. The amplification premixed reaction solution is a commercially available conventional PCR amplification reaction solution. The ABScript III One Step RT-qPCR Probe Kit with UDG V5 (Abclonal) used in this invention can be replaced by any commercially available premixed reaction solution with equivalent reaction capacity.

[0084] Table 6

[0085]

[0086]

[0087] (6) Set the reaction program for the real-time PCR instrument according to Table 7 below.

[0088] Table 7

[0089]

[0090] (7) The Ct values ​​of each target are shown in Table 8 below. All targets involved in this invention can be effectively amplified, and the amplification efficiency meets expectations.

[0091] Table 8

[0092]

[0093]

[0094] Example 3

[0095] High-throughput sequencing was performed on the commercial plasmid from Example 2.

[0096] The specific implementation steps are as follows:

[0097] (1) The concentration was determined using Qubit4.0 and the commercial plasmid (PUC57 Shanghai Sangon Biotech, contract number: GN202318177) was diluted to 1 ng.

[0098] (2) Configure the fragmentation and final repair system according to Table 9 below, and perform fragmentation. The fragmentation and final repair enzyme is a commercially available reagent, and any reagent with the same function can be substituted.

[0099] Table 9

[0100] Components Input Fragmented unrepaired enzyme 8μL plasmid template 10ng Nuclease-free water Add to 50μL

[0101] Set up the PCR program and perform the reaction according to Table 10 below.

[0102] Table 10

[0103] temperature time 30℃ 30min 72℃ 15min

[0104] (3) NGS adapters were added to the unrefined fragmented products. Adapter ligation was performed according to Table 11. The adapters were universal adapters for the Illumina sequencing platform and the indexes required for NGS data splitting. The ligase was commercially available T4 ligase, and any reagent with equivalent efficacy could be substituted.

[0105] Table 11

[0106] Components Input Fragmented final product 50μL Connector (0.05 μmol) 2μL Linker ligase 3μL Connector buffer 25μL

[0107] Table 12

[0108] temperature time 20℃ 30min 12℃ Hold

[0109] (4) The adapter ligation products are purified using nucleic acid purification magnetic beads. The magnetic beads are commercially available nucleic acid adsorption magnetic beads with the ability to adsorb nucleic acids, and any magnetic beads with equivalent effectiveness can be used as a substitute.

[0110] (5) The purified product was amplified into a library. The amplification system was configured according to Table 13, and the PCR program was set according to Table 14. In the amplification system, the amplification primers were universal primers for the Illumina sequencing platform.

[0111] Table 13

[0112] Components Input Purified product 20μL Amplification reaction solution 25μL Amplification primers 5μL

[0113] Table 14

[0114]

[0115] (6) After amplification, the amplification product is purified using the magnetic beads from step (5). After purification, it becomes a library that can be used for high-throughput sequencing.

[0116] (7) The high-throughput sequencing results of the plasmids used in this invention are as follows: Figure 3 As shown, targets in each channel were detected to a certain extent, verifying the effectiveness of the detection results of this invention.

[0117] In summary, this invention, through a primer design approach that combines conservatism and specificity, and based on one-step quantitative real-time PCR technology, enables rapid differentiation of pathogen types in patients. It has a wide target range, high detection efficiency, and ensures that the probe maintains stable binding with the target fragment during primer extension, resulting in high accuracy and effectively avoiding cross-contamination and aerosol contamination.

[0118] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A combination of a primer set and a fluorescent probe set for detecting a pathogenic microorganism, characterized by, The primer set consists of sequences represented by nucleic acid sequences SEQ ID NO. 1-SEQ ID NO. 42; the nucleic acid sequences of the fluorescent probe set consist of sequences represented by SEQ ID NO. 43-SEQ ID NO. 63; The primer set and the fluorescent probe set are configured in the same reaction system, and four fluorescent channels of FAM, VIC, ROX and Cy5 are used to specifically detect and distinguish RNA viruses, fungi, DNA viruses and bacteria respectively, and each fluorescent channel corresponds to the detection result of a type of pathogenic microorganism uniquely; The 5' end of the fluorescent probe set contains a fluorescent group, and the 3' end contains a quenching group; The fluorescent group includes FAM, Cy5, VIC and ROX; The quenching group includes BHQ1, BHQ2 and MGB.

2. The combination of primer sets and fluorescent probe sets for detecting a pathogenic microorganism according to claim 1, wherein The pathogenic microorganism includes any one or a combination of at least two of bacteria, fungi, DNA viruses or RNA viruses.

3. The combination of primer sets and fluorescent probe sets for detecting a pathogenic microorganism according to claim 2, wherein The fungi include any one or a combination of at least two of Cryptococcus fungi, Candida fungi, Aspergillus fungi or Pneumocystis fungi.

4. The combination of primer sets and fluorescent probe sets for detecting pathogenic microorganisms according to claim 2, wherein The DNA virus includes any one or a combination of at least two of human herpes virus I, human herpes virus II, human herpes virus III, human herpes virus IV, human herpes virus V, hepatitis B virus, human parvovirus B19 or all types of adenovirus.

5. The combination of primer sets and fluorescent probe sets for detecting pathogenic microorganisms according to claim 2, wherein The RNA virus includes any one or a combination of at least two of rhinovirus, influenza A virus, influenza B virus, coronavirus 229E, coronavirus OC43, coronavirus HKU1, coronavirus NL63, novel coronavirus or enterovirus.

6. Use of the primer set and the fluorescent probe set for detecting pathogenic microorganisms according to any one of claims 1-5 in the preparation of a product for detecting pathogenic microorganisms.

7. A kit for detecting a pathogenic microorganism, characterized by, The kit comprises the primer set and the fluorescent probe set for detecting pathogenic microorganisms according to any one of claims 1-5.

8. A method for detecting pathogenic microorganisms for non-disease diagnosis and / or treatment purposes, characterized by, The method comprises the following steps: (1) Extracting DNA from the sample to be tested as a template, and performing one-step fluorescent quantitative PCR amplification using the primer set and the fluorescent probe set for detecting pathogenic microorganisms according to any one of claims 1-5; (2) Collecting and detecting the fluorescence signals of each channel of each reaction well in the PCR amplification to generate an amplification curve; (3) Determining whether the sample to be tested contains pathogenic microorganisms according to the Ct values of the fluorescence signals of each channel.

9. The method of claim 8, wherein, The pathogenic microorganism includes any one or a combination of at least two of bacteria, fungi, DNA viruses or RNA viruses.

10. The method of claim 9, wherein, The fungi include any one or a combination of at least two of Cryptococcus fungi, Candida fungi, Aspergillus fungi or Pneumocystis fungi.

11. The method of claim 9, wherein, The DNA virus includes any one or a combination of at least two of human herpes virus I, human herpes virus II, human herpes virus III, human herpes virus IV, human herpes virus V, hepatitis B virus, human parvovirus B19 or all types of adenovirus.

12. The method of claim 9, wherein, The RNA virus includes any one or a combination of at least two of rhinovirus, influenza A virus, influenza B virus, coronavirus 229E, coronavirus OC43, coronavirus HKU1, coronavirus NL63, novel coronavirus or enterovirus.

13. The method of claim 8, wherein, Each reaction well in step (2) contains four fluorescence channels, each corresponding to a type of pathogen.

14. The method of claim 8, wherein, The judgment criteria in step (3) are: The fluorescence channel Ct value for detecting bacteria is <28, which is determined as positive for bacteria; The fluorescence channel Ct value for detecting fungi is <33, which is determined as positive for fungi; The fluorescence channel Ct value for detecting DNA virus is <30, which is determined as positive for DNA virus; The fluorescence channel Ct value for detecting RNA virus is <30, which is determined as positive for RNA virus.

15. The method according to any one of claims 8-14, characterized in that, The sample to be tested includes any one of blood, lung lavage fluid, nasopharyngeal swab, sputum or cerebrospinal fluid.

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