Kit for detecting multiple pathogens in a single tube using the melting point Tm value of PCR amplification products

Abstract

The present invention provides a kit for realizing single-tube detection of multiple pathogens using the melting point Tm value of a PCR amplification product, the kit includes a specific probe and a downstream primer designed for each pathogen to be tested, a part of the specific probe is used as a specific upstream primer, and the Tm value of the specific upstream primer is controlled at 50-70°C, and the Tm value range of the probe is 60-80°C, so as to realize the control of the melting point Tm value of the fluorescent product of each pathogen to be tested, so that the melting point Tm values ​​of the fluorescent products of different pathogens to be tested in the same fluorescent labeling channel can be distinguished by a PCR instrument. The kit has endogenous internal references and exogenous internal references as quality control, respectively, to avoid false negatives of detection. Through the Tm difference of the fluorescently labeled PCR products of different pathogens to be tested and / or different fluorescent labels, multiple detections of multiple pathogens to be tested are realized, the detection accuracy is improved, and time, cost and sample size are saved.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a kit for realizing single-tube detection of multiple pathogens by using the melting point Tm value of a PCR amplification product. Background Art

[0002] Traditional real-time fluorescence PCR technology adds different fluorescent groups to the PCR reaction system and uses the accumulation of fluorescent signals to monitor the entire PCR process in real time. It has the advantages of high sensitivity, high specificity, effective solution to PCR contamination problems, and rapidity, and is currently a widely used method for nucleic acid detection. However, due to the limitations of the channels of fluorescent PCR instruments currently on the market, a single tube can only detect 4-5 targets at most, which cannot meet the clinical needs of simultaneous screening of multiple pathogens or genes related to a certain syndrome.

[0003] At present, common clinical symptoms of pathogenic microorganism infection include respiratory tract infection, bloodstream infection, urogenital tract infection, etc. The pathogenic microorganisms that cause these symptoms are relatively complex, including bacteria, viruses, mycoplasma, chlamydia, fungi, etc., and multiple pathogenic microorganisms need to be detected at the same time, which makes disease diagnosis and treatment extremely difficult. If the disease caused by infection cannot be diagnosed quickly, it can only be treated based on the experience and existing knowledge of clinicians. These treatment methods are often accompanied by the abuse of broad-spectrum antibiotics, causing the emergence of multidrug-resistant bacteria and the occurrence of hospital-acquired infections. The strategy of simultaneous detection of multiple pathogens and the rapid and accurate identification of pathogens are crucial in disease management.

[0004] The traditional culture method is the current gold standard for diagnosis, but this method is slow, cumbersome, unable to perform quantitative tests, and has a high false negative rate. For patients with bloodstream infection that develops into septic shock, their survival time drops sharply in hours if they lack timely treatment. Therefore, we urgently need a method to quickly and accurately identify pathogens. In recent years, with the development of molecular detection methods, methods for rapid diagnosis of BSI pathogens have emerged in an endless stream. These molecular diagnostic techniques are used to supplement the deficiencies of culture and can be divided into detection methods based on pathogen detection in positive blood culture samples and direct detection of pathogens in whole blood. The former includes matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, fluorescence in situ hybridization technology, multiplex PCR technology, DNA microarray technology, etc.; the latter includes real-time quantitative PCR, second-generation sequencing technology, digital PCR technology, etc. The second-generation sequencing technology can quickly sequence all DNA or RNA in the sample, can detect multiple target genes at one time, and has the advantages of high sensitivity and specificity, and small sample size required. However, it has disadvantages such as high testing cost, relatively long cycle, inability to detect drug-resistant genes, and the need for a professional team. In particular, the test report often takes 2 to 3 days. However, it has more advantages in the identification of clinically rare bacteria and the discovery of new strains.

[0005] Given the current clinical demand for simple, rapid, highly sensitive, specific and multi-target detection technologies, and the limitations of existing technologies, which cannot fully meet clinical needs, we urgently need to develop a fast, accurate and low-cost multiplex detection technology. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a kit for realizing single-tube detection of multiple pathogens using the melting point Tm value of PCR amplification products, the kit comprising:

[0007] a. Design a specific probe and a downstream primer for each pathogen to be tested, a part of the specific probe is used as a specific upstream primer, the Tm value of the specific upstream primer is controlled at 50-70°C, and the Tm value range of the probe is 60-80°C, so as to control the melting point Tm value of the fluorescent product of each pathogen to be tested, so that the melting point Tm values ​​of the fluorescent products of different pathogens to be tested in the same fluorescent labeling channel can be distinguished by a PCR instrument; the probe contains at least one RNA base, a fluorescent group is marked on the probe base on the left side of the RNA base close to the 5' end of the probe, and a quenching group is marked on the probe base on the side of the RNA base close to the 3' end of the probe; the melting point Tm value of the probe fragment on the left side of the RNA base is higher than the melting point Tm value of the probe fragment on the right side of the RNA base; the downstream primer and the probe are respectively specifically combined with the respective pathogens to be tested to form a probe-template hybrid double strand, and a DNA-RNA hybrid strand is formed at the RNA base;

[0008] b. a heat-resistant ribonuclease RNaseH, which cuts the RNA base bound to the nucleic acid sequence to be detected, so that the fragment containing the fluorescent group on the left side of the RNA base still remains to form a hybrid chain, while the probe fragment containing the quenching group on the right side of the RNA base is freed;

[0009] c. Nucleic acid polymerase and dNTP. When the fragment containing a fluorescent group on the left side of the RNA base is used as a primer, the polymerase enables the pathogen to be detected to generate a PCR product with a fluorescent label.

[0010] In one embodiment, the kit is a single-tube kit for detecting Candida tropicalis, Candida glabrata, Enterobacter cloacae, Candida albicans, Staphylococcus aureus, Serratia marcescens, Proteus mirabilis, Enterococcus faecalis, and Klebsiella pneumoniae, and their primers and probes are as follows:

[0011]

[0012]

[0013] In one embodiment, the final concentration of the primers for Candida tropicalis, Candida glabrata, Candida albicans, Staphylococcus aureus, Serratia marcescens, Proteus mirabilis, Enterococcus faecalis, and / or Klebsiella pneumoniae is 0.1-0.3 mM and the final concentration of the probe is 0.05-0.2 mM, preferably, the final concentration of the primers for Candida tropicalis is 0.1 mM and the final concentration of the probe is 0.05 mM; and / or, the final concentration of the primers for Candida glabrata is 0.1 mM and the final concentration of the probe is 0.05 mM; and / or Or, the final concentration of the Enterobacter cloacae primer is 0.2mM and the final concentration of the probe is 0.1mM; and / or, the final concentration of the Candida albicans primer is 0.1mM and the final concentration of the probe is 0.05mM; and / or, the final concentration of the Staphylococcus aureus primer is 0.2mM and the final concentration of the probe is 0.2mM; and / or, the final concentration of the Serratia marcescens primer is 0.2mM and the final concentration of the probe is 0.1mM; and / or, the final concentration of the Proteus mirabilis primer is 0.1mM and the final concentration of the probe is 0.05mM; and / or, the final concentration of the Enterococcus faecalis primer is 0.1mM and the final concentration of the probe is 0.05mM; and / or, the final concentration of the Klebsiella pneumoniae primer (KPN-R1) is 0.1mM and the final concentration of the probe is 0.05mM.

[0014] In one embodiment, the kit is a single-tube test kit, which detects Acinetobacter baumannii, Escherichia coli, Staphylococcus epidermidis, Enterococcus faecium, Pseudomonas aeruginosa, Candida krusei, Klebsiella oxytoca, Streptococcus pneumoniae, Candida parapsilosis and Streptococcus agalactiae, and their primers and probes are as follows:

[0015]

[0016]

[0017] In one embodiment, the final concentration of the primers for Acinetobacter baumannii, Escherichia coli, Staphylococcus epidermidis, Enterococcus faecium, Pseudomonas aeruginosa, Candida krusei, Klebsiella oxytoca, Streptococcus pneumoniae, Candida parapsilosis and / or Streptococcus agalactiae is 0.1-0.3 mM and the final concentration of the probe is 0.05-0.2 mM.

[0018] In one embodiment, the final concentration of the primer for Acinetobacter baumannii is 0.1 mM and the final concentration of the probe is 0.1 mM; and / or, the final concentration of the primer for Escherichia coli is 0.1 mM and the final concentration of the probe is 0.1 mM; and / or, the final concentration of the primer for Staphylococcus epidermidis is 0.2 mM and the final concentration of the probe is 0.1 mM; and / or, the final concentration of the primer for Escherichia coli is 0.1 mM and the final concentration of the probe is 0.1 mM; and / or, the final concentration of the Staphylococcus aureus primer is 0.2 mM and the final concentration of the probe is 0.2 mM; and / or, the final concentration of the Pseudomonas aeruginosa primer is 0.1 mM and the final concentration of the probe is 0.1 mM; and / or, the final concentration of the Candida krusei primer is 0.1 mM and the final concentration of the probe is 0.1 mM; and / or, the final concentration of the Klebsiella oxytoca primer is 0.2 mM and the final concentration of the probe is 0.1 mM; and / or, the final concentration of the Streptococcus pneumoniae primer is 0.1 mM and the final concentration of the probe is 0.1 mM, and / or, the final concentration of the Candida parapsilosis primer is 0.1 mM and the final concentration of the probe is 0.1 mM, and / or, the final concentration of the Streptococcus agalactiae primer is 0.2 mM and the final concentration of the probe is 0.2 mM.

[0019] In one embodiment, the dNTP concentration is set to 0.2 mM.

[0020] In one embodiment, the polymerase concentration is 1.0U.

[0021] In one embodiment, a kit for detecting multiple bloodstream infection pathogens is provided, the kit comprising a first tube of reagents of the kit as described in claim 2 and a second tube of reagents of the kit as described in claim 4, an endogenous internal reference quality control is provided in the first tube of reagents, and an exogenous internal reference quality control is provided in the second tube of reagents.

[0022] In one embodiment, the probe primers for the endogenous internal reference and the exogenous internal reference are as follows:

[0023]

[0024] The present invention designs a specific probe and a downstream primer for each target nucleic acid sequence to be detected. The probe contains at least one RNA base. The Tm value of each target nucleic acid sequence to be detected is controlled by controlling the sequence length between the probe and the downstream primer, so that the melting point Tm values ​​of the fluorescent products of different target nucleic acid sequences to be detected in the same fluorescent labeling channel can be distinguished on a fluorescent PCR instrument. The melting point Tm value of the probe fragment on the left side of the 5' end of the RNA base is higher than the melting point Tm value of the probe fragment on the right side of the RNA base. The Tm value of the sequence on the left side of the 5' end of the RNA base and the Tm value of the probe are controlled within a certain range, thereby improving the amplification efficiency and detection sensitivity of the sequence after RNA enzyme cleavage; directly performing melting curve analysis on the specific product to reduce the number of sequences in the amplification system, and at the same time reducing the formation and amplification of sequence dimers by setting different annealing temperatures and extension temperatures (extension temperature is higher than annealing temperature) in the reaction program; having excellent specificity. The main advantages of the present invention are as follows:

[0025] 1). Detect multiple target nucleic acids based on the melting curve of the product, control the Tm value of the sequence on the left side of the 5' end of the RNA base at 50-70°C, and the Tm value range of the probe is 60-80°C. Only the probe and downstream primers need to be designed for the sequence to be tested to complete the detection and amplification, reduce the number of sequences in the amplification system, reduce the formation of polymers between different sequences, and have high sensitivity and specificity.

[0026] 2). In the existing fluorescent quantitative PCR amplification, the annealing temperature and the extension temperature are usually the same temperature. In the present invention, by setting different annealing temperatures and extension temperatures in the reaction program (the extension temperature is higher than the annealing temperature), the formation and amplification of sequence dimers are reduced and the specificity is improved.

[0027] 3). It is conducive to obtaining fluorescently labeled primers by RNaseH digestion, amplifying products with fluorescent groups, and directly performing melting curve analysis on specific products, which has excellent specificity;

[0028] 4). Utilize the different channels of the fluorescence quantitative PCR instrument and the Tm difference two-dimensional parameters of different products to achieve multiple detection of multi-target nucleic acids, improve detection accuracy, save time, cost and sample volume.

[0029] 5). The entire process is performed in a closed tube to avoid amplification contamination.

[0030] In each of the two tubes of detection reagents of the present invention, an internal reference quality control is selected, which are an endogenous internal reference quality control and an exogenous internal reference quality control. The endogenous internal reference can be used to quality control whether the sampling is successful, while the exogenous internal reference can be used to quality control whether the amplification is successful.

[0031] In the present invention, the concentration of primers and probes in the reaction system of the kit is verified to determine the optimal primer and probe concentration for each target. The concentration of dNTPs and Taq polymerase in the reaction system of the kit is verified to determine the optimal dNTP concentration and Taq polymerase concentration.

[0032] In one embodiment, the kit comprises a hot-start DNA polymerase, UDG enzyme, and a thermostable ribonuclease RNaseH.

[0033] In one embodiment, the kit further includes a positive control and a negative control; the positive control is a recombinant plasmid fragment of the constructed target gene to be amplified, and the negative control is RNase-free and DNase-free water.

[0034] The present invention provides a kit with high detection sensitivity and good specificity. Its detection accuracy reaches the quality level of similar products already on the market and can meet the needs of clinical trials. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0036] Figure 1 The result graphs of the detection reagent 1 of the kit of the present invention for detecting each target strain at a concentration of 100 CFU / mL are CTR (represented by A1), CG (represented by A2), CA (represented by A3), and ENC (represented by A4) of the HEX channel; ENF (represented by B1) of the ROX channel; SA (represented by C1) and SMS (represented by C2) of the CY5 channel; and product melting curves of PM (represented by D1) and KPN (represented by D2) of the FAM channel;

[0037] Figure 2 The result graphs of the detection reagent 2 of the kit of the present invention for detecting each target strain at a concentration of 100 CFU / mL are GBS (A1), ABA (A2), and STAE (A3) of the HEX channel; CPA (B1), EFS (B2), and PA (B3) of the ROX channel; CTR (C1), and KLO (C2) of the CY5 channel; and the product melting curve graphs of SP (D1) and ECO (D2) of the FAM channel;

[0038] Figure 3The kit of the present invention detects product melting curves of Streptococcus mitis, Streptococcus pyogenes, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Micrococcus luteus, Rhodococcus equi, Listeria monocytogenes, Acinetobacter junii, Haemophilus parainfluenzae, Neisseria sicca, Pseudomonas fluorescens, Aeromonas hydrophila, Candida albicans, and Legionella pneumophila (derived from 34 national reference products for multiple nucleic acid detection reagents for bacterial and fungal infections). DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with the embodiments. Obviously, the embodiments are only examples and cannot limit the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.

[0040] In the following examples, unless otherwise specified, all methods are conventional in the art. All reagents in the examples are commercially available. Reaction buffer and detection enzyme solution were purchased from a professional raw material company; all primers and probes used were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.

[0041] Identification of primers and probes for multiplex detection of nucleic acids from nineteen bloodstream infection pathogens

[0042] 1. Primer probe sequence screening

[0043] 1.1. Primer probe sequence The present invention analyzes the gene sequences of pathogens such as Pseudomonas aeruginosa (PA), Acinetobacter baumannii (ABA), Klebsiella pneumoniae (KPN), Escherichia coli (ECO), Staphylococcus aureus (SA), Enterobacter cloacae (ENC), Staphylococcus epidermidis (STAE), Candida tropicalis (CTR), Candida krusei (CKR), Candida albicans (CA), Klebsiella oxytoca (KLO), Serratia marcescens (SMS), Proteus mirabilis (PM), Streptococcus pneumoniae (SP), Enterococcus faecalis (ENF), Enterococcus faecium (EFS), Candida parapsilosis (CPA), Candida glabrata (CG) and Streptococcus agalactiae (GBS), designs primers and probes based on the basic principles of primer and probe design using design software, and screens primer and probe combinations for each pathogen target. The primer and probe combinations screened for each pathogen target are shown in Table 1.

[0044] Table 1. Primer probes screened for 19 bloodstream infection pathogen targets

[0045]

[0046]

[0047]

[0048]

[0049]

[0050] 1.2. Template used: low concentration standard plasmid dilutions (1 copy / μL and 10 copies / μL) of each target of 19 bloodstream infection pathogens, human internal reference and exogenous internal reference

[0051] 1.3. Concentration of kit components

[0052] The concentrations of each component are: RNaseH is 20mU, dNTP is 0.2mM, MgSO4 is 3mM, primer concentration is 0.1mM~0.3mM, probe concentration is 0.05mM~0.2mM, and DNA polymerase is 1.0U.

[0053] 1.4. Reaction procedure: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 15 seconds, annealing at 58°C, extension at 72°C, collecting fluorescence during extension, repeat 45 cycles; melting curve analysis at 40-95°C, detecting fluorescence signals every 0.04°C, cooling at 60°C for 1 minute.

[0054] 1.5. Screening results: As shown in Table 2 below, the 21 targets were screened for the primer probes with the best sensitivity and the most suitable Tm values ​​in the reaction solution combination: CTR-P1 / CTR-R1 of Candida tropicalis, CG-P2 / CG-R2 of Candida glabrata, ENC-P1 / ENC-R1 of Enterobacter cloacae, IC6-P2 / IC6-R2 of internal reference (ROX), CA-P3 / CA-R3 of Candida albicans, SA-P1 / SA-R1 of Staphylococcus aureus, SMS- P2 / SMS-R2 of Serratia marcescens, PM-P3 / PM-R3 of Proteus mirabilis, ENF-P2 / ENF-R2 of Enterococcus faecalis, KPN-P1 / KPN-R1 of Klebsiella pneumoniae in reaction solution 1; ABA-P1 / ABA-R1 in reaction solution 2, ECO- The sensitivity of the best primer probe single complex for each target can be amplified to 1 copy / μL.

[0055] Table 2. Results of single-plex validation of primers and probes for screening 21 bloodstream infection pathogens

[0056]

[0057]

[0058] 2. Determination of target combinations in multiplex reactions

[0059] Melting curve experiments are not simply mixing multiple pairs of specific primer probes into a reaction system. In melting curve experiments, the primer probes of each target are required not to bind to areas other than the target fragment on the template DNA, so as to ensure specificity in multiplex detection; therefore, when conducting multiplex experiments, the concentrations of primer probes used for multiple targets also need to be optimized to achieve higher amplification sensitivity.

[0060] 2.1 The concentrations of the components of the multiple reaction solution are as follows:

[0061] The concentrations of each component are: RNaseH is 20mU, dNTP is 0.2mM, MgSO4 is 3mM, primer concentration is 0.1mM~0.3mM, probe concentration is 0.05mM~0.2mM, and DNA polymerase is 1.0U.

[0062] 2.1.1 Screening of primer and probe concentration

[0063] 2.1.1.1 Screening of primer and probe concentration in reaction solution 1:

[0064] When the primer concentration was 0.1mM to 0.3mM and the probe concentration was 0.05mM to 0.2mM, 10 copies / μL of plasmid of each target was tested for comparison: the results are shown in Table 3

[0065] Table 3 Screening of primer concentrations of target probes in reaction solution 1

[0066]

[0067]

[0068] The results showed that when the final concentration of the Candida tropicalis primer (CTR-R1) was 0.1mM and the final concentration of the probe (CTR-P1) was 0.05mM, the Ct value of the detection was the smallest compared with other concentrations, indicating that the detection efficiency was the highest; the primer concentration was 0.1-0.3mM and the probe concentration was 0.05-0.2mM, both of which could meet the detection sensitivity requirements.

[0069] When the final concentration of the Candida glabrata primer (CG-R2) was 0.1mM and the final concentration of the probe (CG-P2) was 0.05mM, the Ct value of the detection was the smallest compared with other concentrations, indicating that the detection efficiency was the highest; the primer concentration was 0.1-0.3mM and the probe concentration was 0.05-0.2mM, both of which could meet the detection sensitivity requirements.

[0070] When the final concentration of the Enterobacter cloacae primer (ENC-R1) was 0.2 mM and the final concentration of the probe (ENC-P1) was 0.1 mM, the Ct value of the detection was the smallest compared with other concentrations, indicating that the detection efficiency was the highest; the primer concentration was 0.1-0.3 mM and the probe concentration was 0.05-0.2 mM, both of which could meet the detection sensitivity requirements.

[0071] When the final concentration of the Candida albicans primer (CA-R3) was 0.1mM and the final concentration of the probe (CA-P3) was 0.05mM, the Ct value of the detection was the smallest compared with other concentrations, indicating that the detection efficiency was the highest; the primer concentration was 0.1-0.3mM and the probe concentration was 0.05-0.2mM, both of which could meet the detection sensitivity requirements.

[0072] When the final concentration of the Staphylococcus aureus primer (SA-R1) is 0.2mM and the final concentration of the probe (SA-P1) is 0.2mM, the Ct value of the detection is the smallest compared with other concentrations, indicating that the detection efficiency is the highest; the primer concentration is 0.1-0.3mM and the probe concentration is 0.05-0.2mM, both of which can meet the detection sensitivity requirements.

[0073] When the final concentration of the Serratia marcescens primer (SMS-R2) was 0.2 mM and the final concentration of the probe (SMS-P2) was 0.1 mM, the Ct value of the detection was the smallest compared with other concentrations, indicating that the detection efficiency was the highest; the primer concentration was 0.1-0.3 mM and the probe concentration was 0.05-0.2 mM, both of which could meet the detection sensitivity requirements.

[0074] When the final concentration of the human genome conserved gene primer (IC6-R2) is 0.2mM and the final concentration of the probe (IC6-P2) is 0.2mM, the Ct value of the detection is the smallest compared with other concentrations, indicating that the detection efficiency is the highest; the primer concentration is 0.1-0.3mM and the probe concentration is 0.05-0.2mM, both of which can meet the detection sensitivity requirements.

[0075] When the final concentration of the Proteus mirabilis primer (PM-R3) was 0.1mM and the final concentration of the probe (PM-P3) was 0.05mM, the Ct value of the detection was the smallest compared with other concentrations, indicating that the detection efficiency was the highest; the primer concentration was 0.1-0.3mM and the probe concentration was 0.05-0.2mM, both of which could meet the detection sensitivity requirements.

[0076] When the final concentration of the Enterococcus faecalis primer (ENF-R2) was 0.1 mM and the final concentration of the probe (ENF-P2) was 0.05 mM, the Ct value of the detection was the smallest compared with other concentrations, indicating that the detection efficiency was the highest; the primer concentration was 0.1-0.3 mM and the probe concentration was 0.05-0.2 mM, both of which could meet the detection sensitivity requirements.

[0077] When the final concentration of Klebsiella pneumoniae primer (KPN-R1) was 0.1mM and the final concentration of probe (KPN-P1) was 0.05mM, the Ct value of the detection was the smallest compared with other concentrations, indicating the highest detection efficiency; the primer concentration was 0.1-0.3mM and the probe concentration was 0.05-0.2mM, both of which could meet the detection sensitivity requirements.

[0078] 2.1.1.2 Screening of primer and probe concentrations in reaction solution 2, see Table 4

[0079] Table 4 Screening of primer concentrations of target probes in reaction solution 2

[0080]

[0081]

[0082] The results showed that when the final concentration of the Acinetobacter baumannii primer (ABA-R1) was 0.1mM and the final concentration of the probe (ABA-P1) was 0.1mM, the Ct value of the detection was the smallest compared with other concentrations, indicating that the detection efficiency was the highest; the primer concentration was 0.1-0.3mM and the probe concentration was 0.05-0.2mM, both of which could meet the detection sensitivity requirements.

[0083] When the final concentration of the Escherichia coli primer (ECO-R2) is 0.1mM and the final concentration of the probe (ECO-P2) is 0.1mM, the Ct value of the detection is the smallest compared with other concentrations, indicating that the detection efficiency is the highest; the primer concentration is 0.1-0.3mM and the probe concentration is 0.05-0.2mM, both of which can meet the detection sensitivity requirements.

[0084] When the final concentration of the exogenous internal reference primer (WNC-R1) was 0.2 mM and the final concentration of the probe (WNC-P1) was 0.2 mM, the Ct value of the detection was the smallest compared with other concentrations, indicating that the detection efficiency was the highest; the primer concentration was 0.1-0.3 mM and the probe concentration was 0.05-0.2 mM, both of which could meet the detection sensitivity requirements.

[0085] When the final concentration of Staphylococcus epidermidis primer (STAE-R3) was 0.2mM and the final concentration of probe (STAE-P3) was 0.1mM, the Ct value of detection was the smallest compared with other concentrations, indicating the highest detection efficiency; the primer concentration was 0.1-0.3mM and the probe concentration was 0.05-0.2mM, both of which could meet the detection sensitivity requirements.

[0086] When the final concentration of the fecal intestinal primer (EFS-R1) is 0.1mM and the final concentration of the probe (EFS-P1) is 0.1mM, the Ct value of the detection is the smallest compared with other concentrations, indicating that the detection efficiency is the highest; the primer concentration is 0.1-0.3mM and the probe concentration is 0.05-0.2mM, both of which can meet the detection sensitivity requirements.

[0087] When the final concentration of Pseudomonas aeruginosa primer (PA-R2) was 0.1mM and the final concentration of probe (PA-P2) was 0.1mM, the Ct value of the detection was the smallest compared with other concentrations, indicating the highest detection efficiency; the primer concentration was 0.1-0.3mM and the probe concentration was 0.05-0.2mM, both of which could meet the detection sensitivity requirements.

[0088] When the final concentration of the Candida krusei primer (CKR-R3) was 0.1mM and the final concentration of the probe (CKR-P3) was 0.1mM, the Ct value of the detection was the smallest compared with other concentrations, indicating that the detection efficiency was the highest; the primer concentration was 0.1-0.3mM and the probe concentration was 0.05-0.2mM, both of which could meet the detection sensitivity requirements.

[0089] When the final concentration of Klebsiella oxytoca primer (KLO-R1) was 0.2mM and the final concentration of probe (KLO-P1) was 0.1mM, the Ct value of detection was the smallest compared with other concentrations, indicating the highest detection efficiency; the primer concentration was 0.1-0.3mM and the probe concentration was 0.05-0.2mM, both of which could meet the detection sensitivity requirements.

[0090] When the final concentration of the Streptococcus pneumoniae primer (SP-R2) is 0.1mM and the final concentration of the probe (SP-P2) is 0.1mM, the Ct value of the detection is the smallest compared with other concentrations, indicating that the detection efficiency is the highest; the primer concentration is 0.1-0.3mM and the probe concentration is 0.05-0.2mM, both of which can meet the detection sensitivity requirements.

[0091] When the final concentration of the Candida parapsilosis primer (CPA-R2) was 0.1 mM and the final concentration of the probe (CPA-P2) was 0.1 mM, the Ct value of the detection was the smallest compared with other concentrations, indicating that the detection efficiency was the highest; the primer concentration was 0.1-0.3 mM and the probe concentration was 0.05-0.2 mM, both of which could meet the detection sensitivity requirements.

[0092] When the final concentration of the Streptococcus agalactiae primer (GBS-R2) is 0.2mM and the final concentration of the probe (GBS-P2) is 0.2mM, the Ct value of the detection is the smallest compared with other concentrations, indicating that the detection efficiency is the highest; the primer concentration is 0.1-0.3mM and the probe concentration is 0.05-0.2mM, both of which can meet the detection sensitivity requirements.

[0093] 2.1.2 Screening of dNTP concentrations The dNTP concentrations were set to 0.1 mM, 0.2 mM, and 0.3 mM, and 10 copies / μL of each target plasmid were tested in two reaction solutions. The results are shown in Table 5-6

[0094] Table 5 Screening of dNTP concentration of reaction solution 1

[0095]

[0096]

[0097] Table 6 Screening of dNTP concentration of reaction solution 2

[0098]

[0099] The results show that when the final dNTP concentration is 0.3mM, the Ct value is 1-2 values ​​behind the final dNTP concentration of 0.2mM; when the final dNTP concentration is 0.1mM, the Ct value is ahead of the other two concentrations, but the blank will be non-specific. The non-specific Ct value of the blank of reaction solution 1 is 40, and the Tm value is 77; the non-specific Ct value of the blank of reaction solution 2 is 39, and the Tm value is 85. By comparison, when the dNTP concentration is 0.2mM, the detected Ct value is the smallest, indicating that the detection efficiency is the highest. 2.1.2 Screening of DNA polymerase concentration The DNA polymerase concentration was set to 0.5U, 1.0U, and 2.0U, and 10 copies / μL of each target plasmid was detected in two reaction solutions, respectively. The results are shown in Table 7-8

[0100] Table 7 Screening of DNA polymerase concentration in reaction solution 1

[0101]

[0102]

[0103] Table 8 Screening of DNA polymerase concentration in reaction solution 2

[0104]

[0105] The results showed that when the final concentration of DNA polymerase was 0.5U, the Ct value of each target was 1-2 behind that of 0.5U; when the final concentration of DNA polymerase was 2.0U, the Ct value was ahead of the other two concentrations, but the blank would be non-specific, the non-specific Ct value of the blank of reaction solution 1 was 41, and the Tm value was 77.51; the non-specific Ct value of the blank of reaction solution 2 was 39.12, and the Tm value was 85.14. By comparison, when the final concentration of DNA polymerase was 1.0U, the Ct value of the detection was the smallest, indicating that the detection efficiency was the highest.

[0106] Example 2 Preparation of Bloodstream Infection Kit

[0107] 1. Kit Components

[0108] 1.1. The kit includes reaction solution buffer 1, reaction solution buffer 2, primer probe Mix 1, primer probe Mix 2, detection enzyme solution, positive control substance, and negative control substance.

[0109] The reaction solution buffer 1 contains Tris-HCl buffer for amplification reaction, potassium chloride, magnesium sulfate, deoxyribonucleotides (5 types, dCTP, dGTP, dATP, dTTP, dUTP), and RNase-free and DNase-free water.

[0110] The reaction solution buffer 2 contains Tris-HCl buffer for amplification reaction, potassium chloride, magnesium sulfate, deoxyribonucleotides (5 types, dCTP, dGTP, dATP, dTTP, dUTP), and RNase-free and DNase-free water.

[0111] Primer probe Mix1 includes primers and probes for Candida tropicalis, Candida glabrata, Enterobacter cloacae, Candida albicans, Enterococcus faecalis, Klebsiella pneumoniae, Staphylococcus aureus, Serratia marcescens, Proteus mirabilis and human-derived internal reference targets.

[0112] Primer probe Mix2 includes primers and probes for Acinetobacter baumannii, Candida parapsilosis, Enterococcus faecium, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Candida krusei, Klebsiella oxytoca, Streptococcus agalactiae and exogenous internal reference targets.

[0113] The detection enzyme solution includes hot-start DNA polymerase, RNaseH2, and UDG enzyme.

[0114] The positive control was a pseudovirus constructed to amplify the target gene, and the negative control was RNase- and DNase-free water.

[0115] 2. Kit detection process of the present invention

[0116] 2.1 Sample DNA extraction

[0117] 2.1.1 Add 3.5 μL proteinase K, 0.5 mL lysis buffer, and 20 μL magnetic beads to a 1.5 ml nuclease-free centrifuge tube (invert before use to mix the beads thoroughly), take 200 μL of sample, and add it to the prepared lysis solution. Vortex thoroughly and lyse for 5 minutes at room temperature, inverting and mixing continuously.

[0118] 2.1.2 Briefly centrifuge to collect the liquid on the tube wall, place the centrifuge tube on the magnetic rack and let it stand for 1 minute. After the magnetic beads are completely adsorbed, use a pipette to aspirate the liquid inside.

[0119] 2.1.3 Remove the centrifuge tube, add 500 μL of washing solution I, and mix by inversion for 1 min.

[0120] 2.1.4 Briefly centrifuge to collect the liquid on the tube wall, place the centrifuge tube on the magnetic rack and let it stand for 1 minute. After the magnetic beads are completely adsorbed, use a pipette to aspirate the liquid inside.

[0121] 2.1.5 Remove the centrifuge tube, add 500 μL of washing solution II, and mix by inversion for 1 min.

[0122] 2.1.6 Briefly centrifuge to collect the liquid on the tube wall, place the centrifuge tube on the magnetic rack and let it stand for 1 minute. After the magnetic beads are completely adsorbed, use a pipette to aspirate the liquid inside. Keep the centrifuge tube on the magnetic rack with the lid open for 3 to 5 minutes and aspirate the liquid at the bottom of the tube.

[0123] 2.1.7 Remove the centrifuge tube, add 60 μL of elution solution, shake and mix, and place in a 55°C water bath for 5 minutes, shaking gently twice during the process. Centrifuge briefly to collect the liquid in the tube, place the centrifuge tube on a magnetic rack, and after the magnetic beads are completely adsorbed, use a pipette to transfer the liquid in the tube to a new nuclease-free centrifuge tube. The resulting liquid is the DNA of the sample to be tested.

[0124] 2.2 Mix the reaction solution buffer 1 or 2, primer probe Mix 1 or 2, detection enzyme solution, and DNA template

[0125]

[0126]

[0127] 2.3 Amplify according to the program in a fluorescent PCR instrument: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 15 seconds, annealing at 58°C, extension at 72°C, collecting fluorescence during extension, repeat 45 cycles; melting curve analysis at 40-95°C, detecting fluorescence signals every 0.04°C, and cooling at 60°C for 1 minute.

[0128] 2.4 After amplification, the results of bloodstream infection pathogens are determined based on the fluorescence curve

[0129] 2.4.1 If there is no obvious amplification curve or the Ct value is greater than 40 in the bloodstream infection pathogen detection, and there is an obvious amplification curve in the internal reference channel detection, and the Ct value is ≤38, the bloodstream infection pathogen nucleic acid test is judged to be negative;

[0130] 2.4.2 The FAM, CY5, ROX, and VIC channels have obvious amplification curves and the detection Ct value is ≤40. The results are judged based on the Tm of the melting curve, and the corresponding channel Tm value target detection is judged to be positive.

[0131] 2.5. Validation of the kit of the present invention on 16 clinical samples

[0132] Table 9 Statistical table of test results of 16 clinical samples (“-” indicates negative test result)

[0133]

[0134]

[0135] Two cases of Streptococcus pneumoniae, six cases of Escherichia coli, two cases of Klebsiella pneumoniae and six cases of Candida tropicalis were detected and all were positive, and the results were accurate.

[0136] Example 3 Sensitivity test of the kit of the present invention

[0137] 1. Preparation of reference materials

[0138] 1.1. Preparation of samples with detection limits for each target pathogen

[0139] The strains of each target pathogen were selected and graded diluted according to the concentration to a concentration of 100 CFU / mL as the detection limit sample.

[0140] 2. Experimental plan

[0141] The test kit of the present invention was used to perform the test according to the method described in Example 2.

[0142] 3. Test results

[0143] The minimum detection limit of each target pathogen is 100 CFU / mL. Figure 1-2 This kit can detect a concentration of 100 CFU / mL.

[0144] Example 4 Specificity test of the kit of the present invention

[0145] The kit of the present invention is used to detect Streptococcus mitis, Streptococcus pyogenes, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Micrococcus luteus, Rhodococcus equi, Listeria monocytogenes, Acinetobacter junii, Haemophilus parainfluenzae, Neisseria sicca, Pseudomonas fluorescens, Aeromonas hydrophila, Candida albicans, and Legionella pneumophila (derived from 34 national reference products for multiple nucleic acid detection reagents for bacterial and fungal infections).

[0146] The test results show that the test kit of the present invention detects Streptococcus mitis, Streptococcus pyogenes, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Micrococcus luteus, Rhodococcus equi, Listeria monocytogenes, Acinetobacter junii, Haemophilus parainfluenzae, Neisseria sicca, Pseudomonas fluorescens, Aeromonas hydrophila, Candida albicans, Legionella pneumophila (derived from 34 kinds of national reference materials for multiple nucleic acid detection reagents for bacterial and fungal infections) and the results are all negative. The melting curve results are shown in Figure 3 .

[0147] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0148] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims. Sequence Listing <110> Jiangsu Hongwei Tes Pharmaceutical Technology Co., Ltd. <120> Kit for detecting multiple pathogens in a single tube using the melting point Tm value of PCR amplification products <130> PF2256 <160> 126 <170> SIPOSequenceListing 1.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <400> 1 ccgttgttga aagttttgac tattgtaa 28 <210> 2 <211> twenty two <212> DNA <213> Artificial Sequence <400> 2 ggtggcggga gcaatcctac cg 22 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <400> 3 gcaattcata tgacgtatcg ca 22 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <400> 4 ttattacaat agtcaccgct tgcaacaacg ga 32 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 gctgacgttc agcgggtagt 20 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <400> 6 accacgatgt atctcataac tgtgacctcg aat 33 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <400> 7 cccaacgaac aaaagaatag tagtaaa 27 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <400> 8 ggagtgcgcg tggagctctc tagtcc 26 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 cgcaggcagg agaaatagca 20 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <400> 10 ggggaggacc agtgtagaca ctcagg 26 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <400> 11 ccaaccgcgc acttaagc 18 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <400> 12 ttctctgctg tgaatgctat ttctcctgcc 30 <210> 13 <211> 15 <212> DNA <213> Artificial Sequence <400> 13 accgtggccg tggca 15 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <400> 14 gcatgtccgc actgtcatgg tcgtgg 26 <210> 15 <211> twenty four <212> DNA <213> Artificial Sequence <400> 15 agttgatcgg gacttcacag taga 24 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <400> 16 gttcaggttc aggtgaaaca gcacgctatc 30 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <400> 17 caaagttgat cgggacttca cagtaga 27 <210> 18 <211> 32 <212> DNA <213> Artificial Sequence <400> 18 agcgtgaagg caacaacctc tactgtgaag tc 32 <210> 19 <211> twenty three <212> DNA <213> Artificial Sequence <400> 19 tcgtattgct caacaccaaa ccc 23 <210> 20 <211> 29 <212> DNA <213> Artificial Sequence <400> 20 cctgtttgag cgtcgtttct ccctcaaac 29 <210> twenty one <211> twenty two <212> DNA <213> Artificial Sequence <400> twenty one gcttaagttc agcgggtagt cc 22 <210> twenty two <211> twenty four <212> DNA <213> Artificial Sequence <400> twenty two cgccgcaagc aatgtttttg gtta 24 <210> twenty three <211> twenty four <212> DNA <213> Artificial Sequence <400> twenty three caatgtgcgt tcaaagattc gatg 24 <210> twenty four <211> 25 <212> DNA <213> Artificial Sequence <400> twenty four taaggcggga tcgctttgac aatgg 25 <210> 25 <211> twenty four <212> DNA <213> Artificial Sequence <400> 25 tttcgtaaat gcacttgctt cagg 24 <210> 26 <211> 38 <212> DNA <213> Artificial Sequence <400> 26 ggttgataca cctgaaacaa agcatcctaa aaaaggtg 38 <210> 27 <211> twenty four <212> DNA <213> Artificial Sequence <400> 27 taaatatacg ctaagccacg tcca 24 <210> 28 <211> 28 <212> DNA <213> Artificial Sequence <400> 28 gaacatgcac cgacaggtga agtcgagt 28 <210> 29 <211> 25 <212> DNA <213> Artificial Sequence <400> 29 tcaattttct ttgcattttc tacca 25 <210> 30 <211> 35 <212> DNA <213> Artificial Sequence <400> 30 agactattat tggccgatac acctgaaaca aagca 35 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <400> 31 cttgcgctgg tcggtcact 19 <210> 32 <211> twenty two <212> DNA <213> Artificial Sequence <400> 32 cgttgccgat gcctggaaag cg 22 <210> 33 <211> 16 <212> DNA <213> Artificial Sequence <400> 33 tgctcttccg gcacgc 16 <210> 34 <211> 28 <212> DNA <213> Artificial Sequence <400> 34 ccagggcgtg gagattatcg acatctcc 28 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <400> 35 ggcaacgcgc tgctcttc 18 <210> 36 <211> twenty two <212> DNA <213> Artificial Sequence <400> 36 acatctcccc aatgggctgc cg 22 <210> 37 <211> 17 <212> DNA <213> Artificial Sequence <400> 37 gggggcggcc agataaa 17 <210> 38 <211> 29 <212> DNA <213> Artificial Sequence <400> 38 aattccgaga cgaccctagc tcctccatg 29 <210> 39 <211> 25 <212> DNA <213> Artificial Sequence <400> 39 tcctttaaat tcccatgaaggatgc 25 <210> 40 <211> 27 <212> DNA <213> Artificial Sequence <400> 40 tgttcgcacc atggcatctc tttagca 27 <210> 41 <211> twenty one <212> DNA <213> Artificial Sequence <400> 41 gacacttaca gctgcccagg t 21 <210> 42 <211> 25 <212> DNA <213> Artificial Sequence <400> 42 agagagtgcc caccgcagtc ccatt 25 <210> 43 <211> twenty one <212> DNA <213> Artificial Sequence <400> 43 gactatattt aggaataatc a 21 <210> 44 <211> 39 <212> DNA <213> Artificial Sequence <400> 44 attaatgggc aaactatcac agtcaccact aatctcacg 39 <210> 45 <211> twenty three <212> DNA <213> Artificial Sequence <400> 45 agatgataat ccggaagatg acg 23 <210> 46 <211> 39 <212> DNA <213> Artificial Sequence <400> 46 gattgaatta cataccgccg tactgtctga aactgaatt 39 <210> 47 <211> 26 <212> DNA <213> Artificial Sequence <400> 47 gcccctgatt ttattaatgg tttgag 26 <210> 48 <211> 40 <212> DNA <213> Artificial Sequence <400> 48 ccttagtact gtctgaaact gaattacaat ctgtgccctc 40 <210> 49 <211> twenty four <212> DNA <213> Artificial Sequence <400> 49 ttgcactaca cagatgcctg attt 24 <210> 50 <211> 26 <212> DNA <213> Artificial Sequence <400> 50 gggaatccat tgccattcct catgga 26 <210> 51 <211> twenty two <212> DNA <213> Artificial Sequence <400> 51 gcaggctgtg ttgagcccgc tt 22 <210> 52 <211> 35 <212> DNA <213> Artificial Sequence <400> 52 cagatagcaa ttggtctctt tcaatcattg cttcg 35 <210> 53 <211> 25 <212> DNA <213> Artificial Sequence <400> 53 gcgttcattc tgattctctc cttta 25 <210> 54 <211> 28 <212> DNA <213> Artificial Sequence <400> 54 tatggataac gtggtgcaac ttgccgac 28 <210> 55 <211> twenty one <212> DNA <213> Artificial Sequence <400> 55 cacttccgga tagccctcca g 21 <210> 56 <211> twenty two <212> DNA <213> Artificial Sequence <400> 56 gggcggcgca cctcttatcc ac 22 <210> 57 <211> twenty three <212> DNA <213> Artificial Sequence <400> 57 tggctcataa gacgtgcttg tag 23 <210> 58 <211> 33 <212> DNA <213> Artificial Sequence <400> 58 cgaaggtgag atgggtgact ctcatatggg tct 33 <210> 59 <211> 18 <212> DNA <213> Artificial Sequence <400> 59 aaaacccacg ctgtcgcc 18 <210> 60 <211> 25 <212> DNA <213> Artificial Sequence <400> 60 tacgtgctgg agggctatcc ggaag 25 <210> 61 <211> twenty four <212> DNA <213> Artificial Sequence <400> 61 ccttcgattt ctgctttagg ggta 24 <210> 62 <211> 27 <212> DNA <213> Artificial Sequence <400> 62 gcacgcccta gaccctcaat atgcacg 27 <210> 63 <211> 30 <212> DNA <213> Artificial Sequence <400> 63 tgaaacaagt aggttatcaa tatctacacc 30 <210> 64 <211> 26 <212> DNA <213> Artificial Sequence <400> 64 acgccctaga ccctcaatat gcacgc 26 <210> 65 <211> twenty three <212> DNA <213> Artificial Sequence <400> 65 caccaagttt gcgtgcatat tga 23 <210> 66 <211> 30 <212> DNA <213> Artificial Sequence <400> 66 tggtacatgt gccttcattg atgctgagca 30 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <400> 67 tcgctacaac gacacctggt 20 <210> 68 <211> 27 <212> DNA <213> Artificial Sequence <400> 68 cactgtgcgc gctttctgct ctgcatt 27 <210> 69 <211> 17 <212> DNA <213> Artificial Sequence <400> 69 gcgcagcggg cgtaaat 17 <210> 70 <211> 31 <212> DNA <213> Artificial Sequence <400> 70 agtttcgctc tcgttttctg cagctctatc a 31 <210> 71 <211> 17 <212> DNA <213> Artificial Sequence <400> 71 cctgtccagc gtgtccg 17 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <400> 72 gcggcgcgta gctctcgacc 20 <210> 73 <211> 28 <212> DNA <213> Artificial Sequence <400> 73 cttggtggtt actcttgaac tctcagtc 28 <210> 74 <211> 34 <212> DNA <213> Artificial Sequence <400> 74 tagttcatgt cccatattatac atggcacaat gtga 34 <210> 75 <211> 20 <212> DNA <213> Artificial Sequence <400> 75 atcgaggccc ttaccaccct 20 <210> 76 <211> 33 <212> DNA <213> Artificial Sequence <400> 76 gggtcaggtc ctattgcaat tacttgaaat gcg 33 <210> 77 <211> 26 <212> DNA <213> Artificial Sequence <400> 77 caccaaacac cagactgtag agaaga 26 <210> 78 <211> 31 <212> DNA <213> Artificial Sequence <400> 78 cgattctgtt tgcacattgc aggtgcttaa t 31 <210> 79 <211> twenty three <212> DNA <213> Artificial Sequence <400> 79 catgtcctgt aagttgcgga atc ​​23 <210> 80 <211> 38 <212> DNA <213> Artificial Sequence <400> 80 cttcccctaa ttcaaacatc tgaacaacaa agtaacag 38 <210> 81 <211> twenty three <212> DNA <213> Artificial Sequence <400> 81 catgtcctgt aagttgcgga atc ​​23 <210> 82 <211> 32 <212> DNA <213> Artificial Sequence <400> 82 cctctcaggt gattcttccc ctaattcaaa ca 32 <210> 83 <211> 29 <212> DNA <213> Artificial Sequence <400> 83 ctaataattt atcatgtccc gtaagttcg 29 <210> 84 <211> 33 <212> DNA <213> Artificial Sequence <400> 84 attggccatg aagaaagtga tgcctctcaa atg 33 <210> 85 <211> 20 <212> DNA <213> Artificial Sequence <400> 85 ttacaagctg ctggtgtgcc 20 <210> 86 <211> 36 <212> DNA <213> Artificial Sequence <400> 86 cttaagtact ggtacataag gaacttgcgg cacacc 36 <210> 87 <211> 25 <212> DNA <213> Artificial Sequence <400> 87 atggaaagac catcctcagg aagta 25 <210> 88 <211> 27 <212> DNA <213> Artificial Sequence <400> 88 tcggataccg caaagagcct tcacatt 27 <210> 89 <211> 25 <212> DNA <213> Artificial Sequence <400> 89 caaacatcgg ataaagcaaa gaacc 25 <210> 90 <211> 28 <212> DNA <213> Artificial Sequence <400> 90 ctgctggtgt gccgcaagtt ccttatgt 28 <210> 91 <211> 17 <212> DNA <213> Artificial Sequence <400> 91 ggatggtcag gccgtcg 17 <210> 92 <211> twenty two <212> DNA <213> Artificial Sequence <400> 92 gccatcgaca acgccctcag ca 22 <210> 93 <211> 18 <212> DNA <213> Artificial Sequence <400> 93 gaccccggca tggctgat 18 <210> 94 <211> twenty three <212> DNA <213> Artificial Sequence <400> 94 atgggcgacg agttgctggc gaa 23 <210> 95 <211> 19 <212> DNA <213> Artificial Sequence <400> 95 atctcgttgc tctcgtgcg 19 <210> 96 <211> 20 <212> DNA <213> Artificial Sequence <400> 96 ttgccgctgg cccattcgct 20 <210> 97 <211> 18 <212> DNA <213> Artificial Sequence <400> 97 cgcttggagc tgcattcc 18 <210> 98 <211> twenty four <212> DNA <213> Artificial Sequence <400> 98 ggaagcagtg aggcccttct gacg 24 <210> 99 <211> twenty three <212> DNA <213> Artificial Sequence <400> 99 tacttgtgcg ctatcggtct ctc 23 <210> 100 <211> twenty three <212> DNA <213> Artificial Sequence <400> 100 gtttgggcat gcagctccaa gcg 23 <210> 101 <211> 17 <212> DNA <213> Artificial Sequence <400> 101 gcatcccacg gggctct 17 <210> 102 <211> 25 <212> DNA <213> Artificial Sequence <400> 102 cggtgtccaa gtcccttgga acagg 25 <210> 103 <211> twenty three <212> DNA <213> Artificial Sequence <400> 103 gtgacggtac agctatcaat ggc 23 <210> 104 <211> 26 <212> DNA <213> Artificial Sequence <400> 104 atctccggac agcgcggtgg ttaaag 26 <210> 105 <211> twenty four <212> DNA <213> Artificial Sequence <400> 105 tagcctttat caagcggata ctgg 24 <210> 106 <211> twenty three <212> DNA <213> Artificial Sequence <400> 106 ctggcgcgtt gttcctgcac agc 23 <210> 107 <211> 20 <212> DNA <213> Artificial Sequence <400> 107 atgtgctggc ttcgatgacg 20 <210> 108 <211> twenty two <212> DNA <213> Artificial Sequence <400> 108 ccaggaatct ccggacagcg cg 22 <210> 109 <211> 25 <212> DNA <213> Artificial Sequence <400> 109 ccacgagtag gagtgatgaa gtaga 25 <210> 110 <211> 28 <212> DNA <213> Artificial Sequence <400> 110 tgctgtagtg tccacccctct gcttccac 28 <210> 111 <211> 26 <212> DNA <213> Artificial Sequence <400> 111 gctttgataa aaggagtcaa ggtagc 26 <210> 112 <211> 35 <212> DNA <213> Artificial Sequence <400> 112 ccgccttcac ttctgtattg tccaaaatct gcttc 35 <210> 113 <211> 26 <212> DNA <213> Artificial Sequence <400> 113 ttggtctata tgtcgagtgt tgctta 26 <210> 114 <211> 27 <212> DNA <213> Artificial Sequence <400> 114 tttccaactt gagagagact tggcggc 27 <210> 115 <211> twenty one <212> DNA <213> Artificial Sequence <400> 115 tactccgcct gtctttcaag c 21 <210> 116 <211> 27 <212> DNA <213> Artificial Sequence <400> 116 agcgtcattt ctccctcaaa ccctcgg 27 <210> 117 <211> twenty two <212> DNA <213> Artificial Sequence <400> 117 ggtttgaggg agaaatgacg ct 22 <210> 118 <211> 34 <212> DNA <213> Artificial Sequence <400> 118 tgaagaacgc agcgaaatgc gataagtaat atga 34 <210> 119 <211> 25 <212> DNA <213> Artificial Sequence <400> 119 ttcatattac gtatcgcatt tcgct 25 <210> 120 <211> 30 <212> DNA <213> Artificial Sequence <400> 120 aacaacggat ctcttggttc tcgcatcgat 30 <210> 121 <211> twenty one <212> DNA <213> Artificial Sequence <400> 121 ggttggcacg caatgaagtc t 21 <210> 122 <211> 35 <212> DNA <213> Artificial Sequence <400> 122 ccggttaatg aggctattac tagtgttgac ccatt 35 <210> 123 <211> 26 <212> DNA <213> Artificial Sequence <400> 123 ttgaatcaac tgaagcaaat ggatct 26 <210> 124 <211> 35 <212> DNA <213> Artificial Sequence <400> 124 ggttagtcaa gcaaatattg atatgggatt tggga 35 <210> 125 <211> twenty four <212> DNA <213> Artificial Sequence <400> 125 caatcacctc tgttgaggct tcta 24 <210> 126 <211> twenty four <212> DNA <213> Artificial Sequence <400> 126 tcattgcgtg ccaaccctga gaca 24

Claims

1. A kit for detecting multiple pathogens in a single tube using the melting point Tm value of a PCR amplification product, characterized in that: The kit comprises: a. Design a specific probe and a downstream primer for each pathogen to be tested, a part of the specific probe is used as a specific upstream primer, the Tm value of the specific upstream primer is controlled at 50-70°C, and the Tm value range of the probe is 60~80°C, so as to control the melting point Tm value of the fluorescent product of each pathogen to be tested, so that the melting point Tm values ​​of the fluorescent products of different pathogens to be tested in the same fluorescent labeling channel can be distinguished by a PCR instrument; the probe contains at least one RNA base, a fluorescent group is labeled on the probe base on the left side of the RNA base close to the 5' end of the probe, and a quenching group is labeled on the probe base on the side of the RNA base close to the 3' end of the probe; the melting point Tm value of the probe fragment on the left side of the RNA base is higher than the melting point Tm value of the probe fragment on the right side of the RNA base; the downstream primer and the probe are specifically combined with the respective pathogens to be tested to form a probe-template hybrid double strand, and a DNA-RNA hybrid strand is formed at the RNA base; b. a heat-resistant ribonuclease RNaseH, wherein the heat-resistant ribonuclease RNaseH cuts the RNA base bound to the nucleic acid sequence to be tested, so that the fragment containing the fluorescent group on the left side of the RNA base still remains to form a hybrid chain, while the probe fragment containing the quenching group on the right side of the RNA base is freed; c. a nucleic acid polymerase and dNTP, wherein the fragment containing a fluorescent group on the left side of the RNA base is used as a primer, and the polymerase generates a fluorescently labeled PCR product of the pathogen to be tested; The kit is a single-tube kit for detecting Candida tropicalis, Candida glabrata, Enterobacter cloacae, Candida albicans, Staphylococcus aureus, Serratia marcescens, Proteus mirabilis, Enterococcus faecalis, and Klebsiella pneumoniae. The primers and probes thereof are as follows: 。 2. The kit according to claim 1, wherein The final concentration of primers for Candida tropicalis, Candida glabrata, Candida albicans, Enterobacter cloacae, Staphylococcus aureus, Serratia marcescens, Proteus mirabilis, Enterococcus faecalis, and / or Klebsiella pneumoniae was 0.1-0.3 mM and the final concentration of probes was 0.05-0.2 mM.

3. The kit according to claim 2, wherein The final concentration of the primer for Candida tropicalis is 0.1 mM and the final concentration of the probe is 0.05 mM; and / or, the final concentration of the primer for Candida glabrata is 0.1 mM and the final concentration of the probe is 0.05 mM; and / or, the final concentration of the primer for Enterobacter cloacae is 0.2 mM and the final concentration of the probe is 0.1 mM; and / or, the final concentration of the primer for Candida albicans is 0.1 mM and the final concentration of the probe is 0.05 mM; and / or, the final concentration of the primer for Staphylococcus aureus is 0. 2mM and the final concentration of the probe is 0.2mM; and / or, the final concentration of the Serratia marcescens primer is 0.2mM and the final concentration of the probe is 0.1mM; and / or, the final concentration of the Proteus mirabilis primer is 0.1mM and the final concentration of the probe is 0.05mM; and / or, the final concentration of the Enterococcus faecalis primer is 0.1mM and the final concentration of the probe is 0.05mM; and / or, the final concentration of the Klebsiella pneumoniae primer is 0.1mM and the final concentration of the probe is 0.05mM.

4. A kit for detecting multiple pathogens in a single tube using the melting point Tm value of a PCR amplification product, characterized in that: The kit comprises: a. Design a specific probe and a downstream primer for each pathogen to be tested, a part of the specific probe is used as a specific upstream primer, the Tm value of the specific upstream primer is controlled at 50-70°C, and the Tm value range of the probe is 60~80°C, so as to control the melting point Tm value of the fluorescent product of each pathogen to be tested, so that the melting point Tm values ​​of the fluorescent products of different pathogens to be tested in the same fluorescent labeling channel can be distinguished by a PCR instrument; the probe contains at least one RNA base, a fluorescent group is labeled on the probe base on the left side of the RNA base close to the 5' end of the probe, and a quenching group is labeled on the probe base on the side of the RNA base close to the 3' end of the probe; the melting point Tm value of the probe fragment on the left side of the RNA base is higher than the melting point Tm value of the probe fragment on the right side of the RNA base; the downstream primer and the probe are specifically combined with the respective pathogens to be tested to form a probe-template hybrid double strand, and a DNA-RNA hybrid strand is formed at the RNA base; b. a heat-resistant ribonuclease RNaseH, wherein the heat-resistant ribonuclease RNaseH cuts the RNA base bound to the nucleic acid sequence to be tested, so that the fragment containing the fluorescent group on the left side of the RNA base still remains to form a hybrid chain, while the probe fragment containing the quenching group on the right side of the RNA base is freed; c. Nucleic acid polymerase and dNTP, when the fragment containing the fluorescent group on the left side of the RNA base is used as a primer, the polymerase enables the pathogen to be tested to generate a fluorescently labeled PCR product; the kit is a single-tube test kit, which detects Acinetobacter baumannii, Escherichia coli, Staphylococcus epidermidis, Enterococcus faecium, Pseudomonas aeruginosa, Candida krusei, Klebsiella oxytoca, Streptococcus pneumoniae, Candida parapsilosis and Streptococcus agalactiae, and their primers and probes are as follows: 。 5. The kit according to claim 4, wherein The final concentration of primers for Acinetobacter baumannii, Escherichia coli, Staphylococcus epidermidis, Enterococcus faecium, Pseudomonas aeruginosa, Candida krusei, Klebsiella oxytoca, Streptococcus pneumoniae, Candida parapsilosis and / or Streptococcus agalactiae was 0.1-0.3 mM and the final concentration of probes was 0.05-0.2 mM.

6. The kit according to claim 5, wherein The final concentration of the primer for Acinetobacter baumannii is 0.1 mM and the final concentration of the probe is 0.1 mM; and / or, the final concentration of the primer for Escherichia coli is 0.1 mM and the final concentration of the probe is 0.1 mM; and / or, the final concentration of the primer for Staphylococcus epidermidis is 0.2 mM and the final concentration of the probe is 0.1 mM; and / or, the final concentration of the primer for Enterococcus faecium is 0.1 mM and the final concentration of the probe is 0.1 mM; and / or, the final concentration of the primer for Pseudomonas aeruginosa is 0.1 mM and the final concentration of the probe is 0.1 mM; And / or, the final concentration of Candida krusei primer is 0.1mM and the final concentration of the probe is 0.1mM; and / or, the final concentration of Klebsiella oxytoca primer is 0.2mM and the final concentration of the probe is 0.1mM; and / or, the final concentration of Streptococcus pneumoniae primer is 0.1mM and the final concentration of the probe is 0.1mM, and / or, the final concentration of Candida parapsilosis primer is 0.1mM and the final concentration of the probe is 0.1mM, and / or, the final concentration of Streptococcus agalactiae primer is 0.2mM and the final concentration of the probe is 0.2mM.

7. The kit according to any one of claims 1 to 6, wherein The dNTP concentration was set to 0.2 mM.

8. The kit according to any one of claims 1 to 6, wherein The polymerase concentration was 1.0U.

9. A kit for detecting multiple bloodstream infection pathogens, characterized in that: The kit comprises a first tube of reagents of the kit according to claim 1 and a second tube of reagents of the kit according to claim 4, wherein an endogenous internal reference quality control is provided in the first tube of reagents, and an exogenous internal reference quality control is provided in the second tube of reagents.

10. The kit according to claim 9, wherein The probe primers for the endogenous internal reference and the exogenous internal reference are as follows: 。

Images