CN117431341A - Primer probe combination, method and application for detecting pathogenic microorganisms - Google Patents

Primer probe combination, method and application for detecting pathogenic microorganisms Download PDF

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Publication number
CN117431341A
CN117431341A CN202311488925.0A CN202311488925A CN117431341A CN 117431341 A CN117431341 A CN 117431341A CN 202311488925 A CN202311488925 A CN 202311488925A CN 117431341 A CN117431341 A CN 117431341A
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detecting
primer
virus
combination
fluorescent
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王胡海灵
赵雅丽
钟佩雯
王婷
林增顺
李英镇
袁剑颖
陈嘉昌
柳俊
胡朝晖
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Guangzhou Jinqirui Biotechnology Co ltd
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Guangzhou Jinqirui Biotechnology Co ltd
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida

Abstract

The invention discloses a primer probe combination, a method and application for detecting pathogenic microorganisms. The nucleic acid sequence of the primer comprises sequences shown in SEQ ID NO.1-SEQ ID NO. 42; the nucleic acid sequence of the fluorescent probe comprises the sequence shown in SEQ ID NO.43-SEQ ID NO. 63. According to the invention, by adopting a primer design mode combining conservation and specificity, a one-step fluorescence quantitative PCR technology is based, so that the pathogen types of pathogens of patients can be rapidly distinguished, the target range is wide, the detection efficiency is high, the probes can be stably combined with target fragments during primer extension, the accuracy is high, and cross contamination and aerosol contamination can be effectively avoided.

Description

Primer probe combination, method and application for detecting pathogenic microorganisms
Technical Field
The invention belongs to the technical fields of biotechnology and molecular biology, and relates to a primer probe combination, a method and application for detecting pathogenic microorganisms.
Background
Infectious diseases are mainly classified into bacterial infection, fungal infection and viral infection (DNA virus and RNA virus). The pathogen detection is an important step in diagnosis and treatment of infectious diseases, different types of pathogen infection have large differences in treatment modes adopted in clinic, for example, bacterial pneumonia needs to be treated by antibiotics, fungal pneumonia is often used as an antifungal drug, and viral pneumonia emphasizes and helps to enhance the autoimmune power of a patient, so that early pathogen type detection can help to formulate a treatment scheme of the patient, and the survival rate of the patient is improved.
The current common detection means of infection pathogen types in clinic are blood routine, serological diagnosis, pathogen microorganism separation culture detection, nucleic acid detection and the like, wherein the blood routine is a method for judging blood conditions and checking diseases by counting the number change and morphological distribution of blood cells, and the patients can be primarily diagnosed to be bacterial infection or virus infection according to the statistical result, but the accuracy of the blood routine is influenced by various physiological factors, and the patients with fungal infection cannot be distinguished, so that the clinical application range is limited. Serological diagnosis refers to a method for detecting corresponding antibodies in serum of a patient by using known antigens, and the method has higher accuracy, but longer clinical turnaround time and influences the diagnosis and treatment efficiency of the patient. The method for separating, culturing and detecting the pathogenic microorganisms is a method for carrying out biological identification after in-vitro culturing on the pathogens, and has low cost, long time consumption and bias. Nucleic acid detection refers to detection of infectious pathogenic microorganisms on a nucleic acid layer, including single or multiple fluorescent quantitative PCR, sequencing and the like, wherein the single or multiple PCR can accurately detect the types of pathogenic species, but has limited coverage range, and a doctor is required to judge the possible infection direction of a patient in advance and then determine the range of PCR targets. Sequencing is currently commonly used in clinic as first generation sequencing, which is usually performed on PCR amplicons of pathogenic microorganisms, and second generation high throughput sequencing (NGS), and can be further classified into tNGS, which is consistent with multiplex PCR, and mNGS, where the range of infection needs to be predetermined. mNGS can carry out full sequencing on infectious pathogens of patients, but has high cost and long clinical turnaround time, and is not suitable for preliminary diagnosis of pathogen types.
In summary, the existing method for detecting pathogenic microorganisms has the problems of limited coverage, need of predetermining an infection range, high cost, long clinical turnaround time, inapplicability to preliminary diagnosis of pathogenic types, and the like. How to provide a method for detecting pathogenic microorganisms with wide detection coverage, high accuracy and high sensitivity has become one of the problems to be solved in the technical fields of biotechnology and molecular biology at present.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides a primer probe combination, a method and application for detecting pathogenic microorganisms, and develops a pathogenic microorganism (bacteria, fungi, DNA viruses and RNA viruses) single-step multiple fluorescence quantitative PCR detection method with wide coverage, short time and high accuracy, thereby relieving the pressure of the current clinical pathogen detection and diagnosis and being capable of assisting doctors in making preliminary diagnosis and treatment schemes for infectious patients in the early stage of admission.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides a primer and a fluorescent probe for detecting pathogenic microorganisms, wherein the nucleic acid sequence of the primer comprises sequences shown in SEQ ID NO.1-SEQ ID NO. 42; the nucleic acid sequence of the fluorescent probe comprises the sequence shown in SEQ ID NO.43-SEQ ID NO. 63.
According to the invention, by adopting a primer design mode combining conservation and specificity, a one-step fluorescence quantitative PCR technology is based, so that the pathogen types of pathogens of patients can be rapidly distinguished, the target range is wide, the detection efficiency is high, the probes can be stably combined with target fragments during primer extension, the accuracy is high, and cross contamination and aerosol contamination can be effectively avoided.
The primer probes designed in the present invention are shown in table 1.
TABLE 1
The one-step multiple fluorescent quantitative PCR adopted by the invention is a multi-target quantitative detection mode based on the birth of the multiple PCR and the one-step fluorescent quantitative PCR, combines the high-efficiency quantitative capability of the one-step fluorescent quantitative PCR and the multi-target detection capability of the multiple PCR, and realizes the amplification and fluorescent detection of multiple target genes or target fragments in a single reaction tube. However, the premise behind achieving such high throughput detection is the precise design between the different target primers and probes to prevent non-specific amplification and primer dimer from occurring, and furthermore, annealing temperatures also need to be tightly controlled.
Preferably, the fluorescent probe comprises a fluorescent group at the 5 'end and a quenching group at the 3' end.
Preferably, the fluorophore comprises any one or a combination of at least two of FAM, cy5, VIC or ROX.
Preferably, the quenching group comprises any one or a combination of at least two of BHQ1, BHQ2 or MGB.
Preferably, the pathogenic microorganism comprises any one or a combination of at least two of bacteria, fungi, DNA viruses or RNA viruses.
Preferably, the fungus comprises any one or a combination of at least two of cryptococcus fungi, candida fungi, aspergillus fungi or pneumospore fungi.
Preferably, the DNA virus comprises any one or a combination of at least two of human herpesvirus I, human herpesvirus II, human herpesvirus III, human herpesvirus IV, human herpesvirus V, hepatitis B virus, human parvovirus B19, or adenovirus all types.
Preferably, the RNA virus comprises 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.
In a second aspect, the invention provides the use of a primer and a fluorescent probe for detecting a pathogenic microorganism according to the first aspect for the preparation of a product for detecting a pathogenic microorganism.
In a third aspect, the present invention provides a kit for detecting a pathogenic microorganism, the kit comprising the primer for detecting a pathogenic microorganism of the first aspect and a fluorescent probe.
In a fourth aspect, the invention provides the primers for detecting pathogenic microorganisms according to the first aspect and the use of fluorescent probes for detecting pathogenic microorganisms.
In a fifth aspect, the present invention provides a method of detecting a pathogenic microorganism, the method comprising the steps of:
(1) Extracting DNA from a sample to be detected as a template, and performing one-step fluorescence quantitative PCR amplification by using the primer and the fluorescent probe for detecting pathogenic microorganisms according to claim 1 or 2;
(2) Collecting and detecting fluorescent signals of each channel of each reaction hole in PCR amplification to generate an amplification curve;
(3) Judging whether the sample to be detected contains pathogenic microorganisms according to the Ct value of the fluorescent signal of each channel.
Preferably, the pathogenic microorganism comprises any one or a combination of at least two of bacteria, fungi, DNA viruses or RNA viruses.
Preferably, the fungus comprises any one or a combination of at least two of cryptococcus fungi, candida fungi, aspergillus fungi or pneumospore fungi.
Preferably, the DNA virus comprises any one or a combination of at least two of human herpesvirus I, human herpesvirus II, human herpesvirus III, human herpesvirus IV, human herpesvirus V, hepatitis B virus, human parvovirus B19, or adenovirus all types.
Preferably, the RNA virus comprises 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.
Preferably, each of the reaction wells in step (2) contains at least four fluorescent channels, each fluorescent channel corresponding to a class of pathogen.
Preferably, each reaction well in step (2) further comprises four fluorescent channels FAM, ROX, VIC and Cy5.
Preferably, the criterion for the determination in step (3) is:
detecting a fluorescence channel Ct value of the bacteria <28, and judging that the bacteria are positive;
detecting the fluorescence channel Ct value of the fungus to be less than 33, and judging that the fungus is positive;
detecting a fluorescence channel Ct value of the DNA virus to be less than 30, and judging that the DNA virus is positive;
the fluorescence channel Ct value of the detected RNA virus is <30, and the positive RNA virus is judged.
Preferably, the sample to be tested includes: any one of blood, alveolar lavage, nasopharyngeal swab, sputum or cerebrospinal fluid.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention realizes rapid differentiation of pathogen types of pathogens of patients based on a one-step fluorescence quantitative PCR technology by a primer design mode combining conservation and specificity, has wide target range coverage, and contains almost all common clinical bacteria, fungi and more than thirty common DNA or RNA viruses. Four pathogen types can be distinguished simultaneously by one-time real-time fluorescence quantitative PCR, which infectious diseases can be known, and whether mixed infection exists or not, so that the detection time is greatly shortened, and the detection efficiency is improved;
(2) The Tm values of all the primers are close to each other and are between 56 and 60 ℃, and the Tm values of all the probes are between 65 and 70 ℃ and higher than 7 to 10 ℃ of the primers, so that the probes can be ensured to keep stable combination with target fragments during primer extension;
(3) The PCR program comprises an amplification system and a reverse transcription process, realizes the whole process by a single tube, does not need to independently carry out reverse transcription of RNA, does not need additional tube opening and pipetting operations, simplifies the operation process, and can effectively avoid cross contamination and aerosol contamination.
Drawings
FIG. 1 is a diagram showing the peak pattern of an amplified product of a primer designed according to the present invention;
FIG. 2 is a graph showing the peak detection of amplification products of poorly performing primers;
FIG. 3 is a graph showing the RPM profile of the target sequence after high throughput sequencing of commercial plasmids.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
And verifying the amplification effect of the primer.
The amplification ability of 21 pairs of primers in total from SEQ ID No.1 to SEQ ID No.63 was verified (experiments include spare primers in the early design).
Each target primer was amplified and detected using a commercial plasmid (PUC 57 Shanghai Biotechnology, contract number: GN 202318177) containing the target sequence as a template.
The primers used in the examples include all the primer sequences of Table 1 and primer sequences not included in Table 1 which are less effective.
The amplification reaction solution used in example 1 is a commercially available universal premixed reaction solution, and the reaction solution comprises Taq enzyme, KCl and MgCl 2 dNTP. Wherein the dNTPs comprise dATP, dGTP, dCTP, dTTP.
The specific procedure of example 1 is as follows:
(1) Each primer was diluted to a working concentration of 10. Mu. Mol/L according to the instructions of the synthesis provider.
(2) The commercial plasmid was subjected to concentration measurement using a spectrophotometer, and diluted to 0.2 ng/. Mu.L.
(3) The PCR reaction system was prepared according to the following Table 2, and the amplification premix was a commercially available conventional PCR amplification reaction, KAPA HIFI Hot Start PCR Mix (Roche) was used in the present invention, and the commercially available premix having the polymerase chain reaction capability was replaced.
TABLE 2
Component (A) Volume (mu L)
Amplification premix reaction (2 Xmix) 12.5
Target index F 0.75
Target index primer R 0.75
Template (commercial plasmid nucleic acid) 2
Nuclease-free water Supplement to 25
(4) The amplification reaction was performed according to the PCR instrument program set up in table 3 below.
TABLE 3 Table 3
(5) The experimental target and theoretical fragment information is shown in Table 4, and amplified fragments were detected using a Qsep-100 fully automated nucleic acid analyzer.
TABLE 4 Table 4
(6) As shown in FIG. 1, the specificity of the amplified products of Q2-Q22 is strong, and the peak tip and other miscellaneous peaks are avoided.
(7) As shown in Table 5 below, sequences with low GC content and possibly dimer structure with other primers were detected, and as a result, as shown in FIG. 2, part of the sequences could not be amplified, part of the sequences had hetero-peaks or dimers, and then, the target probe combinations with poor effects were subjected to one-step multiple qPCR amplification, and as a result, as shown in FIG. 3, the primer probe combinations of the targets were proved to have poor effects, and the sequences in Table 5 were changed to the corresponding sequences in Table 1.
TABLE 5
Example 2
And (3) performing multiplex fluorescence quantitative PCR target amplification detection by a one-step method.
The commercial plasmid (PUC 57 Shanghai Biotechnology, contract number: GN 202318177) is used as a template, and the primer probe designed by the invention is used for one-step multiplex fluorescence quantitative PCR detection.
The one-step multiplex fluorescent quantitative reaction solution used in example 2 was a commercially available universal premix reaction solution.
The specific procedure of example 2 is as follows:
(1) Each primer was diluted to a working concentration of 10. Mu. Mol/L according to the instructions of the synthesis provider.
(2) Each probe was diluted to a working concentration of 10. Mu. Mol/L according to the instructions of the synthesis provider.
(3) The commercial plasmid was subjected to concentration measurement using a spectrophotometer, and diluted to 0.2 ng/. Mu.L.
(4) A one-step multiplex fluorescent quantitative PCR reaction system was prepared according to Table 6 below, and the amplification premix was a commercially available conventional PCR amplification reaction, and the commercially available premix having the same reaction capacity as ABScript IIIOne Step RT-qPCR Probe Kit with UDG V (Abclonal) was used in the present invention.
TABLE 6
(6) The reaction procedure of the fluorescent quantitative PCR apparatus was set according to Table 7 below
TABLE 7
(7) The Ct value information of each target is shown in the following table 8, the targets related to the invention can be effectively amplified, and the amplification efficiency accords with expectations.
TABLE 8
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Example 3
The commercial plasmids of example 2 were subjected to high throughput sequencing.
The specific implementation steps are as follows:
(1) The concentration was determined using Qubit4.0 and the commercial plasmid (PUC 57 Shanghai Biotechnology, contract number: GN 202318177) was diluted to 1ng.
(2) The last fragmentation system was configured to fragment according to table 9 below. The fragmented end repair enzyme is a commercial reagent, and the reagent with the same efficacy can be replaced.
TABLE 9
Component (A) Input amount
Fragmentation end repair enzyme 8μL
Plasmid template 10ng
Nuclease-free water Is added to 50 mu L
The PCR procedure was set up and the reaction was performed according to the following Table 10.
Table 10
Temperature (temperature) Time
30℃ 30min
72℃ 15min
(3) NGS linker addition was performed on the fragmented end repair product and linker ligation was performed as follows in table 11. The linker is an index required by the universal linker of the illuminea sequencing platform and the NGS data splitting. The ligase is commercial T4 ligase, and reagents with the same efficacy can be replaced.
TABLE 11
Component (A) Input amount
Fragmentation end repair products 50μL
Joint (0.05 mu mol) 2μL
Joint ligase 3μL
Joint connection buffer 25μL
Table 12
Temperature (temperature) Time
20℃ 30min
12℃ Hold
(4) The adaptor-ligated product was purified using nucleic acid purification magnetic beads. The magnetic beads are commercially available nucleic acid adsorption magnetic beads, have the capability of adsorbing nucleic acids, and have the same effect, and the magnetic beads can be replaced.
(5) Library amplification was performed on the purified products, the amplification system was configured as in Table 13, and the PCR procedure was set as in Table 14 below. In the amplification system, the amplification primer is a universal primer of an illuminea sequencing platform library.
TABLE 13
Component (A) Input amount
Purification of the product 20μL
Amplification reaction solution 25μL
Amplification primers 5μL
TABLE 14
(6) And (3) purifying the amplified product by using the magnetic beads in the step (5) after amplification, and obtaining the library capable of performing high-throughput sequencing after purification.
(7) The high-throughput sequencing result of the plasmid used in the invention is shown in figure 3, and each channel target is detected to a certain extent, so that the validity of the detection result of the invention is verified.
In conclusion, the invention realizes rapid differentiation of pathogen types of pathogens of patients based on a one-step fluorescence quantitative PCR technology by a primer design mode combining conservation and specificity, has wide target range coverage and high detection efficiency, ensures that a probe can be stably combined with a target fragment during primer extension, has high accuracy, and can effectively avoid cross contamination and aerosol contamination.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims (10)

1. A primer and a fluorescent probe for detecting pathogenic microorganisms, wherein the nucleic acid sequence of the primer comprises sequences shown in SEQ ID NO.1-SEQ ID NO. 42; the nucleic acid sequence of the fluorescent probe comprises the sequence shown in SEQ ID NO.43-SEQ ID NO. 63.
2. The primer and fluorescent probe according to claim 1, wherein the fluorescent probe has a fluorescent group at the 5 'end and a quenching group at the 3' end;
preferably, the fluorophore comprises any one or a combination of at least two of FAM, cy5, VIC or ROX;
preferably, the quenching group comprises any one or a combination of at least two of BHQ1, BHQ2 or MGB.
3. The primer and fluorescent probe for detecting a pathogenic microorganism according to claim 1 or 2, wherein the pathogenic microorganism comprises any one or a combination of at least two of bacteria, fungi, DNA viruses or RNA viruses;
preferably, the fungus comprises any one or a combination of at least two of cryptococcus fungi, candida fungi, aspergillus fungi or pneumospore fungi;
preferably, the DNA virus comprises any one or a combination of at least two of human herpesvirus I, human herpesvirus II, human herpesvirus III, human herpesvirus IV, human herpesvirus V, hepatitis B virus, human parvovirus B19, or adenovirus all;
preferably, the RNA virus comprises 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.
4. Use of a primer and a fluorescent probe for detecting a pathogenic microorganism according to any one of claims 1-3 in the preparation of a product for detecting a pathogenic microorganism.
5. A kit for detecting a pathogenic microorganism, comprising the primer for detecting a pathogenic microorganism according to any one of claims 1 to 3 and a fluorescent probe.
6. Use of a primer and a fluorescent probe for detecting a pathogenic microorganism according to any one of claims 1 to 3 for detecting a pathogenic microorganism.
7. A method of detecting a pathogenic microorganism, the method comprising the steps of:
(1) Extracting DNA from a sample to be detected as a template, and performing one-step fluorescence quantitative PCR amplification by using the primer and the fluorescent probe for detecting pathogenic microorganisms according to claim 1 or 2;
(2) Collecting and detecting fluorescent signals of each channel of each reaction hole in PCR amplification to generate an amplification curve;
(3) Judging whether the sample to be detected contains pathogenic microorganisms according to the Ct value of the fluorescent signal of each channel.
8. The method of claim 7, wherein the pathogenic microorganism comprises any one or a combination of at least two of bacteria, fungi, DNA viruses, or RNA viruses;
preferably, the fungus comprises any one or a combination of at least two of cryptococcus fungi, candida fungi, aspergillus fungi or pneumospore fungi;
preferably, the DNA virus comprises any one or a combination of at least two of human herpesvirus I, human herpesvirus II, human herpesvirus III, human herpesvirus IV, human herpesvirus V, hepatitis B virus, human parvovirus B19, or adenovirus all;
preferably, the RNA virus comprises 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.
9. The method of claim 7 or 8, wherein each of the reaction wells in step (2) comprises at least four fluorescent channels, each fluorescent channel corresponding to a class of pathogens;
preferably, the four fluorescent channels include: FAM, ROX, VIC and Cy5.
Preferably, each reaction well in step (2) further comprises four fluorescent channels FAM, ROX, VIC and Cy 5;
preferably, the criterion for the determination in step (3) is:
detecting a fluorescence channel Ct value of the bacteria <28, and judging that the bacteria are positive;
detecting the fluorescence channel Ct value of the fungus to be less than 33, and judging that the fungus is positive;
detecting a fluorescence channel Ct value of the DNA virus to be less than 30, and judging that the DNA virus is positive;
the fluorescence channel Ct value of the detected RNA virus is <30, and the positive RNA virus is judged.
10. The method according to claims 7-9, wherein the sample to be tested comprises: any one of blood, alveolar lavage, nasopharyngeal swab, sputum or cerebrospinal fluid.
CN202311488925.0A 2023-11-09 2023-11-09 Primer probe combination, method and application for detecting pathogenic microorganisms Pending CN117431341A (en)

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