CN117210586A - Primer probe combination and kit for detecting 9 respiratory tract infection pathogenic bacteria and application of primer probe combination and kit - Google Patents
Primer probe combination and kit for detecting 9 respiratory tract infection pathogenic bacteria and application of primer probe combination and kit Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of in-vitro diagnostic reagent detection, in particular to a primer probe combination, a kit and application thereof for detecting 9 respiratory tract infection pathogens, wherein the kit comprises 9 respiratory tract pathogen amplification primers and fluorescent probe combinations, and the 9 respiratory tract pathogen amplification reactions comprise fluorescent probes, primers, enzyme mixed solution, PCR reaction buffer and the like of a plurality of different pathogen targets, so that whether pertussis bacillus, klebsiella pneumoniae, pseudomonas aeruginosa, streptococcus pneumoniae, stenotrophomonas maltophilia, legionella pneumophila, haemophilus influenzae, group A streptococcus and methicillin-resistant staphylococcus aureus exist can be detected in the same reaction system; the kit also comprises a positive reference substance and a negative reference substance, thereby monitoring the accuracy of PCR detection. The kit for detecting 9 respiratory tract infection pathogens disclosed by the invention has the advantages that the high specificity is kept, the detection sensitivity is obviously improved, and the kit is suitable for rapidly detecting respiratory tract pathogens.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnostic reagent detection, in particular to a primer probe combination for detecting 9 respiratory tract infection pathogenic bacteria, a kit and application thereof.
Background
Respiratory tract infections are caused by a variety of viral and bacterial pathogens, and respiratory diseases are the third leading cause of death worldwide, secondary to cardiovascular and cancer, according to statistics from the U.S. health metric and assessment institute, and pose a serious threat to human health safety and socioeconomic development. Respiratory tract infection refers to the infection of respiratory systems such as nasal cavity, throat, trachea and bronchus of human body by pathogens, and is divided into upper respiratory tract infection and lower respiratory tract infection. Wherein, 1) the upper respiratory tract infection is usually acute upper respiratory tract infection, which refers to the general expression of the acute inflammation of nasal cavity and throat, and is mostly caused by virus infection. 2) Lower respiratory tract infection including acute bronchitis, chronic bronchitis, pneumonia, bronchiectasis, etc. is caused by virus, bacteria, mycoplasma, chlamydia, legionella, etc. microorganisms, and its control should follow the principle of prevention, accurate diagnosis and timely treatment, and the pathogen must be clearly caused to infection during treatment to select effective medicines and treatment methods. Because the clinical symptoms and signs of various respiratory tract pathogen infections are similar, the respiratory tract pathogen infections are mostly manifested as fever, cough or headache, and the like, but the pathological course and the treatment method of the respiratory tract pathogen infections can be obviously different, the respiratory tract pathogen detection reagent can rapidly and accurately detect and identify pathogenic microorganisms, and the treatment method and the medication scheme are determined, so that the respiratory tract pathogen infection detection reagent has important clinical significance.
Major clinical detection projects in respiratory infectious disease progression include pathogen culture, antigen detection, inflammatory marker detection, nucleic acid detection, igM antibody detection, and IgG antibody detection. In recent years, nucleic acid molecule detection is a key technology for detecting respiratory pathogens rapidly due to the characteristics of high specificity, high sensitivity, simplicity, convenience and rapidness, and can be used for screening and diagnosing diseases and providing accurate and timely information, thereby affecting the control of epidemic diseases in administrative and medical departments. However, the common fluorescent quantitative qPCR detection platform can detect 4-5 pathogens at most at the same time, and the technical problem that more than 4-5 pathogens are difficult to detect at the same time exists, so that how to detect more pathogens at the same time on the common qPCR detection platform and improving the detection efficiency is a technical problem which needs to be solved urgently.
Disclosure of Invention
The invention aims to solve the technical problem that a fluorescent quantitative qPCR detection platform is difficult to detect 4-5 pathogens simultaneously, and provides a nucleic acid detection kit for detecting 9 pathogens simultaneously based on a common qPCR detection platform, which can comprehensively monitor the pathogens of the respiratory tract so as to save detection resources. The kit can detect 9 respiratory pathogens including pertussis Bacillus (BP), klebsiella Pneumoniae (KP), pseudomonas Aeruginosa (PA), streptococcus Pneumoniae (SP), stenotrophomonas maltophilia (Sm), legionella Pneumophila (LP), haemophilus influenzae (Hi), group A Streptococcus (GAS) and methicillin-resistant staphylococcus aureus (MRSA) simultaneously.
In order to solve the problems, the invention provides the following technical scheme:
first aspect:
the invention provides a primer probe combination for detecting 9 respiratory tract infection pathogenic bacteria, which comprises the following components:
the BP-F forward primer for detecting the Bordetella Pertussis (BP) is shown as SEQ ID NO.1, the BP-R reverse primer is shown as SEQ ID NO.2, and the BP-P probe is shown as SEQ ID NO. 3;
the KP-F forward primer for detecting Klebsiella Pneumoniae (KP) is shown as SEQ ID NO.4, KP-R reverse primer is shown as SEQ ID NO.5, and KP-P probe is shown as SEQ ID NO. 6;
the forward primer of PA-F for detecting Pseudomonas Aeruginosa (PA) is shown as SEQ ID NO.7, the reverse primer of PA-R is shown as SEQ ID NO.8, and the PA-P probe is shown as SEQ ID NO. 9;
the SP-F forward primer for detecting Streptococcus Pneumoniae (SP) is shown as SEQ ID NO.10, the SP-R reverse primer is shown as SEQ ID NO.11, and the SP-P probe is shown as SEQ ID NO. 12;
the Sm-F forward primer for detecting the stenotrophomonas maltophilia (Sm) is shown as SEQ ID NO.13, the Sm-R reverse primer is shown as SEQ ID NO.14, and the Sm-P probe is shown as sequence SEQ ID NO. 15;
the forward primer of LP-F for detecting Legionella Pneumophila (LP) is shown as SEQ ID NO.16, the reverse primer of LP-R is shown as SEQ ID NO.17, and the probe of LP-P is shown as SEQ ID NO. 18;
the Hi-F forward primer for detecting haemophilus influenzae (Hi) is shown as SEQ ID NO.19, the Hi-R reverse primer is shown as SEQ ID NO.20, and the Hi-P probe is shown as SEQ ID NO. 21;
the forward primer of GAS-F for detecting Group A Streptococcus (GAS) is shown as SEQ ID NO.22, the reverse primer of GAS-R is shown as SEQ ID NO.23, and the GAS-P probe is shown as SEQ ID NO. 24;
MRSA-F forward primer for detecting methicillin-resistant staphylococcus aureus (MRSA) is shown as SEQ ID NO.25, MRSA-R reverse primer is shown as SEQ ID NO.26, and MRSA-P probe is shown as SEQ ID NO. 27.
Second aspect:
the invention provides a kit comprising the primer probe combination for detecting 9 respiratory tract infection pathogenic bacteria.
Further, the kit comprises a reaction solution, an enzyme mixed solution, a positive control substance and a negative quality control substance; the reaction liquid comprises a reaction premix, a primer pair and a fluorescent probe; the reaction premix comprises 1 XPCR reaction buffer and RNase free H2O; the enzyme mixture comprises 1U/. Mu.LTaq DNA polymerase, 0.2U/. Mu.LUDG enzyme and 1U/. Mu.L reverse transcriptase; the positive control contains 9 pathogens and RP gene sequences; the negative control is TE Buffer.
Further, the reaction liquid A, the reaction liquid B and the reaction liquid C are respectively. The three reaction liquids are mutually independent and are not mixed, and different fluorescent groups are arranged in each group of reaction liquids aiming at fluorescent probes of different pathogens to distinguish, so that the aim of simultaneously detecting respiratory pathogens in 9 is fulfilled.
Furthermore, the reaction solution A, the reaction solution B and the reaction solution C all comprise RP-F forward primer, RP-R reverse primer and RP-P fluorescent probe, the human Ribonuclease P (RNase P, RP) is an enzyme commonly existing in cells of various tissues and organs of a human body, and the RNase P can be used as an internal reference gene for detecting RNA of a human sample and monitoring the acquisition, transportation, extraction and amplification processes of the sample to be detected so as to avoid false negative of detection results. Wherein the forward primer of RP-F is shown as SEQ ID NO.28, the reverse primer of RP-R is shown as SEQ ID NO.29, and the fluorescent probe of RP-P is shown as SEQ ID NO. 30.
Preferably, the reaction solution A comprises a reaction premix, a BP-F forward primer, a BP-R reverse primer, a BP-P fluorescent probe, a KP-F forward primer, a KP-R reverse primer, a KP-P fluorescent probe, a PA-F forward primer, a PA-R reverse primer, a PA-P fluorescent probe, a RP-F forward primer, a RP-R reverse primer, and a RP-P fluorescent probe;
preferably, the reaction solution B comprises a reaction premix, SP-F forward primer, SP-R reverse primer, SP-P fluorescent probe, sm-F forward primer, sm-R reverse primer, sm-P fluorescent probe, LP-F forward primer, LP-R reverse primer, LP-P fluorescent probe, RP-F forward primer, RP-R reverse primer, RP-P fluorescent probe;
preferably, the reaction solution C comprises a reaction premix, a Hi-F forward primer, a Hi-R reverse primer, a Hi-P fluorescent probe, a GAS-F forward primer, a GAS-R reverse primer, a GAS-P fluorescent probe, a MRSA-F forward primer, a MRSA-R reverse primer, a MRSA-P fluorescent probe, a RP-F forward primer, a RP-R reverse primer, and a RP-P fluorescent probe;
further, the fluorescent probe is marked with a fluorescent group at the 5 'end and a quenching group at the 3' end, wherein the fluorescent group is selected from any one of FAM, ROX, CY and VIC, and the quenching group is BHQ1 or BHQ2.
Third aspect:
the invention provides application of the kit in the second aspect in detection/auxiliary detection of 9 respiratory tract infection pathogens.
The detection principle is as follows:
if the sample to be detected contains BP in the qPCR reaction process, the labeled FAM probe of the A tube filled with the reaction liquid A generates a fluorescence signal; if the sample to be detected contains KP, the labeled ROX probe of the A tube generates a fluorescent signal; if the sample to be detected contains PA, the labeled CY5 probe of the A tube generates a fluorescent signal; if the sample to be detected contains SP, the labeled FAM probe of the B pipe filled with the reaction liquid B generates a fluorescence signal; if the sample to be detected contains Sm, the labeled ROX probe of the B tube generates a fluorescent signal; if the sample to be detected contains LP, the labeled CY5 probe of the B tube generates a fluorescent signal; if the sample to be detected contains Hi, generating a fluorescent signal by a marked FAM probe of a C pipe filled with a reaction liquid C; if the sample to be detected contains GAS, the labeled ROX probe of the C tube generates a fluorescent signal; if the sample to be tested contains MRSA, the labeled CY5 probe of the C tube generates a fluorescent signal. Furthermore, when the A, B, C three tubes each contain an internal reference gene, the labeled VIC probe should generate a fluorescent signal.
Further, the qPCR reaction procedure was as follows: reverse transcription at 50℃for 20min,1 cycle; pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15s, annealing at 60℃for 30s, and fluorescence was collected for 40 cycles. Further, the fluorescence detection channel comprises FAM, ROX, CY and VIC.
The invention has the following advantages:
1. aiming at the technical problem that a fluorescent quantitative qPCR detection platform in the prior art is difficult to detect more than 4-5 pathogens, the invention provides a nucleic acid detection kit capable of simultaneously, specifically and sensitively detecting 9 respiratory pathogens based on a common qPCR detection platform, and the respiratory pathogens can be comprehensively monitored so as to save detection resources, the detection method is simple and rapid, the accuracy of detection results is high, and the stability and specificity of the kit are good;
2. the kit for detecting 9 respiratory tract infection pathogens comprises amplification primers and fluorescent probe combinations of 9 respiratory tract pathogens. Wherein, 9 respiratory tract pathogens amplification reactions contain fluorescent probes, primers, enzyme mixed solution, PCR reaction buffer and the like of a plurality of different pathogen targets, and whether 9 pathogens exist in pertussis Bacillus (BP), klebsiella Pneumoniae (KP), pseudomonas Aeruginosa (PA), streptococcus Pneumoniae (SP), stenotrophomonas maltophilia (Sm), legionella Pneumophila (LP), haemophilus influenzae (Hi), group A Streptococcus (GAS) and methicillin-resistant staphylococcus aureus (MRSA) can be detected in the same reaction system, and the kit also comprises human conserved gene RP fragments, positive control and negative control, thereby monitoring the accuracy of PCR detection.
3. The kit for detecting 9 respiratory tract infection pathogens disclosed by the invention has the advantages of keeping high specificity, obviously improving the detection sensitivity, being suitable for rapid detection of respiratory tract pathogens, having high efficiency, reliability and simplicity and being suitable for popularization and application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is apparent that the drawings in the following description are only one embodiment of the present invention, and that other embodiments of the drawings may be derived from the drawings provided without inventive effort for a person skilled in the art.
Fig. 1: BP detection result diagram of the invention;
fig. 2: KP detection result diagram of the invention;
fig. 3: the PA detection result diagram of the invention;
fig. 4: SP detection result diagram of the invention;
fig. 5: sm detection result diagram of the invention;
fig. 6: LP detection result graphs of the present invention;
fig. 7: hi detection result diagram of the invention;
fig. 8: the GAS detection result diagram of the invention;
fig. 9: MRSA detection result diagram of the invention;
fig. 10: the detection results of 9 pathogenic bacteria of the positive control product are shown in the specification;
fig. 11: RP detection results of the positive control of the invention;
fig. 12: the negative control detection result of the invention;
fig. 13: the cross reaction detection result graph of the invention.
Detailed Description
The invention is further illustrated by the following examples in conjunction with the accompanying drawings:
example 1
The kit for simultaneously detecting 9 respiratory tract infection pathogens comprises: reaction solution A, reaction solution B, reaction solution C, enzyme mixed solution, positive control substance and negative quality control substance:
the reaction solution A comprises a reaction premix, a BP-F forward primer, a BP-R reverse primer, a BP-P fluorescent probe, a KP-F forward primer, a KP-R reverse primer, a KP-P fluorescent probe, a PA-F forward primer, a PA-R reverse primer and a PA-P fluorescent probe; the reaction solution B comprises a reaction premix, an SP-F forward primer, an SP-R reverse primer, an SP-P fluorescent probe, an Sm-F forward primer, an Sm-R reverse primer, an Sm-P fluorescent probe, an LP-F forward primer, an LP-R reverse primer and an LP-P fluorescent probe; the reaction solution C comprises a reaction premix, a Hi-F forward primer, a Hi-R reverse primer, a Hi-P fluorescent probe, a GAS-F forward primer, a GAS-R reverse primer, a GAS-P fluorescent probe, a MRSA-F forward primer, a MRSA-R reverse primer and a MRSA-P fluorescent probe; the reaction solution A, the reaction solution B and the reaction solution C also respectively comprise RP-F forward primer, RP-R reverse primer and RP-P fluorescent probe.
The reaction premix comprises 1 XPCR reaction buffer and RNase free H 2 O, the concentration of each primer pair and the fluorescent probe is 100-200nmol/L; the enzyme mixture comprises 1U/. Mu.LTaq DNA polymerase, 0.2U/. Mu.L UDG enzyme and 1U/. Mu.L reverse transcriptase; the positive control contains 9 pathogens and RP gene sequences; the negative control is TE Buffer.
Detecting whether pertussis Bacillus (BP), klebsiella Pneumoniae (KP), pseudomonas Aeruginosa (PA), streptococcus Pneumoniae (SP), stenotrophomonas maltophilia (Sm), legionella Pneumophila (LP), haemophilus influenzae (Hi), group A Streptococcus (GAS) and methicillin-resistant staphylococcus aureus (MRSA) exist in three pipes of the same reaction system A, B, C; the 5 'end of the fluorescent probe is marked with a fluorescent group, the 3' end is marked with a quenching group,
the tube A is BP, KP, PA and RP, the 5 '-end fluorescent groups of the fluorescent probe are respectively marked FAM, ROX, cy and VIC, and the 3' -end quenching groups are respectively marked BHQ1, BHQ2 and BHQ1;
the B tube is SP, sm, LP and RP, the 5 '-end fluorescent groups of the fluorescent probe are respectively marked FAM, ROX, cy and VIC, and the 3' -end quenching groups are respectively marked BHQ1, BHQ2 and BHQ1;
the C tube is Hi, GAS, MRSA and RP, the 5 '-end fluorescent groups of the fluorescent probe are respectively marked FAM, ROX, cy and VIC, and the 3' -end quenching groups are respectively marked BHQ1, BHQ2 and BHQ1.
The reaction solution A is arranged in the pipe A, the reaction solution B is arranged in the pipe B, the reaction solution C is arranged in the pipe C, and internal reference control is a human conserved gene RP fragment and is marked with VIC. The internal reference control can display the occurrence of false negative and is used for monitoring the collection, transportation, extraction and amplification processes of the sample to be detected.
Example 2
The detection is carried out on a common qPCR detection platform, and the steps are as follows:
step S1, reagent preparation
All reagents were first removed from the refrigerator and equilibrated to room temperature. The PCR reaction liquid preparation standard is as follows: mu.L of the reaction solution A/B/C and the n.times.1.5. Mu.L of the LMRMP enzyme mixture were each added to a 1.5mL centrifuge tube. The tube was shaken and mixed for several seconds and centrifuged for several seconds.
And (5) subpackaging the PCR reaction liquid. The PCR reaction solution was dispensed into PCR thin-walled reaction tubes at 20. Mu.L per tube.
Step S2, extracting nucleic acid: the nasopharynx swab positive sample to be detected (shown in table 4) is subjected to nucleic acid extraction synchronously with the positive control substance and the negative control substance, specifically, a nucleic acid extraction or purification reagent of Qingdao Hantang biotechnology limited company is used, and a nucleic acid extraction or purification reagent instruction book of Qingdao Hantang biotechnology limited company is operated and referred;
step S3, sample adding: taking out the PCR reaction tube containing the PCR reaction liquid in the step S1, adding 5 mu L of the nucleic acid extracted in the step S2, the positive reference substance and the negative reference substance respectively, covering a tube cover, oscillating uniformly, and then performing instantaneous centrifugation at 3000rpm/min for 20S;
step S4, amplification detection: placing the PCR reaction tube obtained in the step S3 into a fluorescent PCR amplification instrument for amplification detection, wherein the reaction program is set as shown in Table 2;
TABLE 2 fluorescent amplificator reaction procedure
Step S5, judging the result: and judging the sample detection result according to the judgment standard of the table 3.
TABLE 3 criterion
The test results of pertussis Bacillus (BP), klebsiella Pneumoniae (KP), pseudomonas Aeruginosa (PA), streptococcus Pneumoniae (SP), stenotrophomonas maltophilia (Sm), legionella Pneumophila (LP), haemophilus influenzae (Hi), group A Streptococcus (GAS), methicillin-resistant staphylococcus aureus (MRSA) and positive and negative controls are detected and analyzed, and the positive and negative controls meet the requirements, so that the test is effective. BP, KP, PA, SP, sm, LP, hi, GAS, MRSA is detected on the corresponding target, and samples containing other pathogenic bacteria and negative quality control substances are not amplified, so that the detection kit and the detection method have good specificity (the result is shown in Table 4).
Table 4 sample test table
Example 3 sensitivity detection
Homemade standard 10 of 9 targets 8 Gradient dilution to 10 copies/mL 5 copy/mL, 10 4 copy/mL, 10 3 copy/mL was used as template. The negative samples were nasopharyngeal swab samples from healthy volunteers. The diluted template and negative sample were amplified using the kit described in example 1, and the amplification conditions were the same as those in example 1, and were determined based on the result determination criteria in example 1, and the results of the 9 target detection sensitivities are shown in table 5. As can be seen from Table 5, when the pathogen template is 10 3 The kit of example 1 can still be used as a template for amplification at copy/mL, and therefore, the kit of the invention can still be used for detection at low concentration of template, and thus has high sensitivity. Sample concentration of 10 4 The results of the detection of pathogenic bacteria at copy/mL are shown in FIGS. 1 to 9. Meanwhile, the kit provided by the invention has no cross reaction between primer pairs, and has high sensitivity consistent with that of common qPCR.
Table 59 respiratory tract infection pathogenic bacteria nucleic acid detection sensitivity results using the kit
Example 4 specific assay
The concentration of partially inactivated human pathogens including influenza A virus, influenza B virus, human coronavirus, cytomegalovirus, respiratory syncytial virus, human parainfluenza virus, adenovirus, EB virus, novel coronavirus, middle east respiratory syndrome symptom virus, and streptococcus pneumoniae deoxyribonucleic acid liquid indoor quality control (Guangzhou bond biosciences) is 10 6 copy/mL, performing specific detection, wherein the detection process is as shown in example 1, and the results show that other sample results except the positive control product are positive resultsNegative, the invention was demonstrated to have no cross-reaction with other pathogen nucleic acids in this example (see FIG. 13).
The present invention has been described above by way of example, but the present invention is not limited to the above-described embodiments, and any modifications or variations based on the present invention fall within the scope of the present invention.
Claims (10)
1. A primer probe combination for detecting 9 respiratory tract infection pathogens, comprising:
the forward primer for detecting the bordetella pertussis is shown as SEQ ID NO.1, the reverse primer is shown as SEQ ID NO.2, and the probe is shown as SEQ ID NO. 3;
the forward primer for detecting klebsiella pneumoniae is shown as SEQ ID NO.4, the reverse primer is shown as SEQ ID NO.5, and the probe is shown as SEQ ID NO. 6;
the forward primer for detecting pseudomonas aeruginosa is shown as SEQ ID NO.7, the reverse primer is shown as SEQ ID NO.8, and the probe is shown as SEQ ID NO. 9;
the forward primer for detecting streptococcus pneumoniae is shown as SEQ ID NO.10, the reverse primer is shown as SEQ ID NO.11, and the probe is shown as SEQ ID NO. 12;
the forward primer for detecting the stenotrophomonas maltophilia is shown as SEQ ID NO.13, the reverse primer is shown as SEQ ID NO.14, and the probe is shown as SEQ ID NO. 15;
the forward primer for detecting Legionella pneumophila is shown as SEQ ID NO.16, the reverse primer is shown as SEQ ID NO.17, and the probe is shown as SEQ ID NO. 18;
the forward primer for detecting the haemophilus influenzae is shown as a sequence SEQ ID NO.19, the reverse primer is shown as a sequence SEQ ID NO.20, and the probe is shown as a sequence SEQ ID NO. 21;
the forward primer for detecting the streptococcus A is shown as SEQ ID NO.22, the reverse primer is shown as SEQ ID NO.23, and the probe is shown as SEQ ID NO. 24;
the forward primer for detecting methicillin-resistant staphylococcus aureus is shown as SEQ ID NO.25, the reverse primer is shown as SEQ ID NO.26, and the probe is shown as SEQ ID NO. 27.
2. A kit comprising the primer probe combination of claim 1 for detecting 9 respiratory tract infection pathogens.
3. The kit according to claim 2, wherein the kit comprises a reaction solution, an enzyme mixed solution, a positive control and a negative control; the reaction liquid comprises a reaction premix, a primer pair and a fluorescent probe.
4. The kit according to claim 3, wherein three kinds of reaction solutions are provided, namely reaction solution A, reaction solution B and reaction solution C.
5. The kit according to claim 4, wherein the reaction solution A comprises a reaction pre-mixture solution, a BP-F forward primer, a BP-R reverse primer, a BP-P fluorescent probe, a KP-F forward primer, a KP-R reverse primer, a KP-P fluorescent probe, a PA-F forward primer, a PA-R reverse primer, a PA-P fluorescent probe, a RP-F forward primer, a RP-R reverse primer, and a RP-P fluorescent probe.
6. The kit of claim 4, wherein the reaction solution B comprises a reaction pre-mixture solution, SP-F forward primer, SP-R reverse primer, SP-P fluorescent probe, sm-F forward primer, sm-R reverse primer, sm-P fluorescent probe, LP-F forward primer, LP-R reverse primer, LP-P fluorescent probe, RP-F forward primer, RP-R reverse primer, RP-P fluorescent probe.
7. The kit of claim 4, wherein the reaction solution C comprises a reaction pre-mixture solution, a Hi-F forward primer, a Hi-R reverse primer, a Hi-P fluorescent probe, a GAS-F forward primer, a GAS-R reverse primer, a GAS-P fluorescent probe, a MRSA-F forward primer, a MRSA-R reverse primer, a MRSA-P fluorescent probe, a RP-F forward primer, a RP-R reverse primer, and a RP-P fluorescent probe.
8. The kit of claim 3, wherein the fluorescent probe is labeled at the 5 'end with a fluorescent group and at the 3' end with a quenching group, wherein the fluorescent group is selected from any one of FAM, ROX, CY and VIC, and the quenching group is BHQ1 or BHQ2.
9. Use of a kit according to any one of claims 2-8 for the detection/assisted detection of 9 respiratory tract infection pathogens.
10. The use according to claim 9, characterized in that the qPCR reaction procedure is as follows: reverse transcription at 50℃for 20min,1 cycle; pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15s, annealing at 60℃for 30s, and fluorescence was collected for 40 cycles, and the fluorescence detection channel included FAM, ROX, CY, VIC.
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