A primer-probe combination, kit and application thereof for detecting bloodstream infection pathogens

Abstract

The present invention provides a primer-probe combination, a kit, and its application for detecting bloodstream infection pathogens. Specifically, the present invention provides a primer-probe combination for detecting bloodstream infection pathogens, the primer-probe group comprising: (a) upstream and downstream primers and probes for detecting coagulase-negative Staphylococci as shown in SEQ ID NOs: 7 to 9; (b) upstream and downstream primers and probes for detecting Escherichia coli as shown in SEQ ID NOs: 1 to 3; (c) upstream and downstream primers and probes for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 4 to 6; and (d) upstream and downstream primers and probes for detecting Staphylococcus aureus as shown in SEQ ID NOs: 10 to 12. The primer-probe combination of the present invention can be used to achieve rapid, highly sensitive, and highly specific detection of bloodstream infection pathogens.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnosis, and in particular to a primer-probe combination, a kit and applications thereof for detecting bloodstream infection pathogens. Background Art

[0002] A bloodstream infection (BSI) refers to the presence of pathogenic microorganisms in a patient's blood. Pathogens that cause BSIs include bacteria, fungi, viruses, and parasites, and can lead to bacteremia, sepsis, and severe cases, shock, disseminated intravascular coagulation (DIC), multiple organ failure, and even death.

[0003] The morbidity and mortality rates of bloodstream infections are rising annually due to factors such as the increase in invasive procedures and the increased use of broad-spectrum antibiotics and corticosteroids. The prognosis after a bloodstream infection can be poor, leading to prolonged hospitalization and an increased financial burden on patients. The earlier the bloodstream infection pathogen is identified and the appropriate antimicrobial treatment is selected, the patient's mortality rate decreases by approximately 7%. Therefore, prompt and rapid identification of the pathogen causing a bloodstream infection is crucial.

[0004] For a long time, there have been many problems in the clinical diagnosis and treatment of BSI, such as early rapid diagnosis and precise treatment, which urgently need to be improved. The current detection methods for BSI mainly include: (1) blood culture, but it has disadvantages such as low detection rate, long detection time, contamination and high false positive rate; (2) MALD-TOF MS technology, which has good identification efficiency for Gram-negative bacteria (>90% consistent with subsequent culture results), but still needs to be used with caution when identifying Gram-positive bacteria (about 80% consistent with subsequent culture results); (3) Next-Generation Sequencing (NGS), which is expensive and therefore not suitable for the rapid detection required by general bloodstream infection patients. In addition, NGS requires the use of PCR to enrich the DNA of the sample to be tested, so there will be amplification bias for some segments, which may lead to missed detection and insufficient accuracy.

[0005] Therefore, it is necessary to develop a method and kit for detecting bloodstream infection that is rapid, highly specific, highly sensitive, and has strong anti-interference ability. Summary of the Invention

[0006] The present invention provides a method and a kit for detecting bloodstream infection rapidly, with high specificity, high sensitivity and strong anti-interference ability.

[0007] In a first aspect of the present invention, a primer-probe combination (or primer-probe composition) for detecting bloodstream infection pathogens is provided, wherein the primer-probe combination (or primer-probe composition) comprises:

[0008] (a) Upstream and downstream primers and probes for detecting coagulase-negative staphylococci as shown in SEQ ID NOs: 7 to 9.

[0009] In another preferred embodiment, the primer-probe combination further comprises a primer-probe combination selected from the following group:

[0010] (b) upstream and downstream primers and probes for detecting Escherichia coli as shown in SEQ ID NOs: 1 to 3;

[0011] (c) upstream and downstream primers and probes for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 4 to 6;

[0012] (d) Upstream and downstream primers and probes for detecting Staphylococcus aureus as shown in SEQ ID NOs: 10-12.

[0013] (e) Any two or more of the above (b) to (d).

[0014] In a preferred aspect, the present invention provides a primer-probe combination for detecting bloodstream infection pathogens, the primer-probe combination comprising:

[0015] (a) upstream and downstream primers and probes for detecting coagulase-negative Staphylococci as shown in SEQ ID NOs: 7 to 9;

[0016] (b) upstream and downstream primers and probes for detecting Escherichia coli as shown in SEQ ID NOs: 1 to 3;

[0017] (c) upstream and downstream primers and probes for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 4 to 6; and

[0018] (d) Upstream and downstream primers and probes for detecting Staphylococcus aureus as shown in SEQ ID NOs: 10-12.

[0019] In another preferred embodiment, the primer-probe combination further includes primers and probes for detecting other bloodstream infection pathogens.

[0020] In another preferred embodiment, the primer-probe combination and the primer-probe composition have the same meaning and can be used interchangeably.

[0021] In another preferred embodiment, the primer-probe combination further comprises:

[0022] (e) Upstream and downstream primers and probes for detecting the internal control as shown in SEQ ID NOs: 13-15.

[0023] In another preferred embodiment, the primers and probes used to detect the internal reference are designed for a specific conserved region of the human EGFR gene.

[0024] In another preferred embodiment, the coagulase-negative staphylococci include: Staphylococcus hominis, Staphylococcus capitis, Staphylococcus simulans, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus caprae, Staphylococcus lugdunensis, Staphylococcus worderi, Staphylococcus xylosus and Staphylococcus sciuri.

[0025] In another preferred embodiment, the fluorescent groups of the probes in the combination are different from each other and do not interfere with each other.

[0026] In this context, "different and non-interfering" means that the fluorescent groups used in each probe in the composition are different and do not affect each other's detection, that is, they can be detected using different channels. For example, FAM, HEX, ROX, CY5, and Q705 can be used. These groups have different absorbance values ​​and can be used in different channels, thus preventing interference.

[0027] In another preferred embodiment, the fluorescent reporter group of the coagulase-negative Staphylococcus probe is ROX; the fluorescent reporter group of the Escherichia coli probe is FAM; the fluorescent reporter group of the Klebsiella pneumoniae probe is HEX; and the fluorescent reporter group of the Staphylococcus aureus probe is CY5.

[0028] In another preferred embodiment, the fluorescent reporter group of the probe used to detect the internal reference is Q705.

[0029] In another preferred embodiment, the 3' end of the probe further has a fluorescence quenching group.

[0030] In another preferred embodiment, the fluorescence quenching group is selected from the following group: BHQ1, BHQ2, and BHQ3.

[0031] In another preferred embodiment, the quencher group at the 3' end of the Escherichia coli probe and the Klebsiella pneumoniae probe is BHQ1; the quencher group at the 3' end of the coagulase-negative Staphylococcus probe and the Staphylococcus aureus probe is BHQ2.

[0032] In another preferred embodiment, the quenching group at the 3' end of the internal reference is BHQ3.

[0033] In another preferred embodiment, each component of the primer-probe combination is present in a separate package.

[0034] In another preferred embodiment, the components of the primer-probe combination are present in the same package.

[0035] In another preferred embodiment, the components of the combination are present in a mixed form.

[0036] In another preferred embodiment, the upstream and downstream primers and probes for detecting coagulase-negative staphylococci are designed for a specific conserved region of the sodA gene of coagulase-negative staphylococci.

[0037] In another preferred embodiment, the upstream and downstream probes for detecting Escherichia coli are designed for a specific conserved region of the ydhQ gene of Escherichia coli.

[0038] In another preferred embodiment, the upstream and downstream primers and probes for detecting Klebsiella pneumoniae are designed for a specific conserved region of the phoE gene of Klebsiella pneumoniae.

[0039] In another preferred embodiment, the upstream and downstream primers and probes for detecting Staphylococcus aureus are designed for a specific conserved region of the BioY gene of Staphylococcus aureus.

[0040] In another preferred embodiment, the 5' end of the probe sequence is connected to a fluorescent reporter gene, and the 3' end is connected to a fluorescent quencher group.

[0041] In a second aspect of the present invention, there is provided a use of the primer-probe combination according to the first aspect of the present invention for preparing a kit for detecting bloodstream infection pathogens; wherein the pathogens include: coagulase-negative Staphylococcus, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus.

[0042] In another preferred embodiment, the coagulase-negative staphylococci include: Staphylococcus hominis, Staphylococcus capitis, Staphylococcus mimicus, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus caprae, Staphylococcus lugdunensis, Staphylococcus worderi, Staphylococcus xylosus and Staphylococcus squirri.

[0043] In a third aspect of the present invention, a kit for detecting bloodstream infection pathogens is provided, wherein the kit comprises the primer-probe combination according to the first aspect of the present invention.

[0044] In another preferred embodiment, the kit further comprises a negative control product and a positive control product.

[0045] In another preferred embodiment, the negative control product is at least one of DEPC-H2O, physiological saline, internal standard gene pseudovirus, and normal human leukocyte genomic DNA.

[0046] In another preferred embodiment, the negative control product is normal human leukocyte genomic DNA (tgDNA).

[0047] In another preferred embodiment, the copy number concentration of the tgDNA is 1000 copies / μL.

[0048] In another preferred embodiment, the positive quality control product is at least one of a plasmid fragment or a DNA fragment of coagulase-negative Staphylococcus, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus.

[0049] In another preferred embodiment, the positive control product is a mixture of coagulase-negative Staphylococcus, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus genomes.

[0050] In another preferred embodiment, the copy number of all target sites in the positive quality control product is 50 copies / μL.

[0051] In another preferred embodiment, the primer-probe combination further comprises:

[0052] (e) Upstream and downstream primers and probes for detecting the internal control as shown in SEQ ID NOs: 13-15.

[0053] In another preferred embodiment, the primers and probes used to detect the internal reference are designed for a specific conserved region of the human EGFR gene.

[0054] In another preferred embodiment, the kit further comprises a PCR reaction solution.

[0055] In another preferred embodiment, the PCR reaction solution includes dNTP, PCR buffer, Mg 2+ and at least one of a DNA polymerase.

[0056] In another preferred embodiment, the DNA polymerase is 5×HS Taq Buffer with Mg 2+ reaction system.

[0057] In another preferred embodiment, the kit further comprises an instruction manual, which indicates that the kit is used to detect bloodstream infection pathogens.

[0058] In another preferred embodiment, the kit further comprises a blank control, which is ultrapure water.

[0059] In a fourth aspect of the present invention, there is provided a use of the primer-probe combination according to the first aspect of the present invention or the kit according to the third aspect of the present invention for preparing a reagent for detecting bloodstream infection pathogens.

[0060] In another preferred embodiment, the detection comprises the following steps:

[0061] (i) Extracting free DNA from the sample to be tested;

[0062] (ii) performing digital PCR on the free DNA obtained in step (i) using the primer-probe combination described in the first aspect of the present invention or the kit described in the third aspect of the present invention;

[0063] (iii) Read the fluorescence data and calculate the copy concentration of the target sequence to obtain the detection result.

[0064] In another preferred embodiment, in step (iii), the determination of the test result is as follows:

[0065] (1) If there are ≥3 positive droplets and the cluster is consistent with the positive control, it is considered a positive result and the infectious pathogen and copy number are directly reported;

[0066] (2) If there are no positive droplets and the clusters are consistent with the negative control, it indicates a negative result and is reported as not detected;

[0067] (3) If there are 1 or 2 positive droplets, it is indicated as a gray area and retesting is recommended; if the retest still shows less than 3 positive droplets, it is determined to be a negative result and reported as not detected.

[0068] In another preferred embodiment, the sample to be tested is selected from the group consisting of whole blood, plasma, serum, or a combination thereof.

[0069] In a fifth aspect of the present invention, a method for detecting bloodstream infection pathogens is provided, comprising the following steps:

[0070] (i) Extracting free DNA from the sample to be tested;

[0071] (ii) performing digital PCR on the free DNA obtained in step (i) using the primer-probe combination described in the first aspect of the present invention or the kit described in the third aspect of the present invention;

[0072] (iii) Read the fluorescence data and calculate the copy concentration of the target sequence to obtain the detection result.

[0073] In another preferred embodiment, in step (iii), the determination of the test result is as follows:

[0074] (1) If there are ≥3 positive droplets and the cluster is consistent with the positive control, it is considered a positive result and the infectious pathogen and copy number are directly reported;

[0075] (2) If there are no positive droplets and the clusters are consistent with the negative control, it indicates a negative result and is reported as not detected;

[0076] (3) If there are 1 or 2 positive droplets, it is indicated as a gray area and retesting is recommended; if the retest still shows less than 3 positive droplets, it is determined to be a negative result and reported as not detected.

[0077] In another preferred embodiment, the sample to be tested is selected from the group consisting of whole blood, plasma, serum, or a combination thereof.

[0078] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0079] In another preferred embodiment, the method is an in vitro method.

[0080] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 The figure shows the detection results of Escherichia coli positive detected by the primer-probe combination of the present invention.

[0082] Figure 2 The figure shows the positive detection results of Klebsiella pneumoniae detected using the primer-probe combination of the present invention.

[0083] Figure 3 The figure shows the positive detection results of Staphylococcus aureus detected using the primer-probe combination of the present invention.

[0084] Figure 4 The figure shows the detection results of coagulase-negative staphylococci detected using the primer-probe combination of the present invention.

[0085] Figure 5 The figure shows the detection result of the internal control (EGFR) positive detected using the primer-probe combination of the present invention.

[0086] Figure 6 Shown are the concentration results for amplification of the E. coli target gene using a multiplex PCR panel.

[0087] Figure 7 Concentration results for singleplex amplification of the target gene in E. coli are shown.

[0088] Figure 8 Shown are the concentration results for amplification of the Klebsiella pneumoniae target gene using a multiplex PCR panel.

[0089] Figure 9 Concentration results for singleplex amplification of the K. pneumoniae target gene are shown.

[0090] Figure 10 Shown are the concentration results for amplification of the target gene of coagulase-negative staphylococci using a multiplex PCR panel.

[0091] Figure 11 Concentration results are shown for singleplex amplification of the target gene from coagulase-negative staphylococci.

[0092] Figure 12 Shown are the concentration results for amplification of the S. aureus target gene using a multiplex PCR panel.

[0093] Figure 13 Concentration results for singleplex amplification of the S. aureus target gene are shown.

[0094] Figure 14 The detection sensitivity of Escherichia coli in Example 2 is shown.

[0095] Figure 15 The detection sensitivity of Klebsiella pneumoniae in Example 2 is shown.

[0096] Figure 16 The detection sensitivity of coagulase-negative staphylococci (Staphylococcus hominis) in Example 2 is shown.

[0097] Figure 17 The detection sensitivity of coagulase-negative staphylococci (Staphylococcus capitis) in Example 2 is shown.

[0098] Figure 18 The detection sensitivity of coagulase-negative staphylococci (mimicking staphylococci) in Example 2 is shown.

[0099] Figure 19 The detection sensitivity of coagulase-negative staphylococci (Staphylococcus saprophyticus) in Example 2 is shown.

[0100] Figure 20 The detection sensitivity of coagulase-negative Staphylococcus (Staphylococcus epidermidis) in Example 2 is shown.

[0101] Figure 21 The detection sensitivity of coagulase-negative staphylococci (hemolytic Staphylococci) in Example 2 is shown.

[0102] Figure 22 The detection sensitivity of coagulase-negative staphylococci (Staphylococcus caprae) in Example 2 is shown.

[0103] Figure 23 The detection sensitivity of coagulase-negative staphylococci (Staphylococcus lugdunensis) in Example 2 is shown.

[0104] Figure 24 The detection sensitivity of the coagulase-negative staphylococcus (Staphylococcus worderi) in Example 2 is shown.

[0105] Figure 25The detection sensitivity of the coagulase-negative staphylococcus (Staphylococcus xylosus) in Example 2 is shown.

[0106] Figure 26 The detection sensitivity of the coagulase-negative staphylococci (S. sciuri) in Example 2 is shown.

[0107] Figure 27 The detection sensitivity of Staphylococcus aureus in Example 2 is shown.

[0108] Figure 28 The specific detection results of the mixed pathogens in Example 3 are shown. DETAILED DESCRIPTION

[0109] After extensive and intensive research and extensive screening, the inventors unexpectedly developed primers and probes that can be used to rapidly, specifically, and sensitively detect bloodstream infection pathogens. These include coagulase-negative Staphylococci, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Experiments conducted by the present invention demonstrated no difference in amplification concentration between singleplex and multiplex PCR, indicating no interaction between the primers and probes that would reduce PCR amplification efficiency. Furthermore, the digital PCR method of the present invention achieves a minimum detection sensitivity of 5 copies / reaction and excellent specificity. This is the basis for the completion of the present invention.

[0110] the term

[0111] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0112] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0113] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range and, where appropriate, fractional values ​​thereof (e.g., tenths and hundredths of an integer).

[0114] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0115] bloodstream infection

[0116] Bloodstream infection (BSI) refers to an infection caused by pathogenic microorganisms entering the bloodstream. The main pathogens include bacteria, fungi, and viruses. BSI is characterized by high morbidity, mortality, high treatment costs, and difficulty in diagnosis.

[0117] Currently, blood culture is the gold standard for laboratory diagnosis of BSI, but its positive rate is only around 10%, and it takes at least 2-3 days to report culture results. There are also some molecular detection methods, such as BioMérieux's FilmArray BCID and LμMinex's Verigene Gram+ BC and Verigene Gram- BC, but these molecular detection methods are all based on positive blood culture cultures. There are also some mNGS-based detection methods in China, but mNGS detection takes 1-2 days, is costly, complex to operate, and requires professional bioinformatics analysts, making it difficult to widely implement in medical institutions. None of these methods have obtained registration certificates from the National Medical Products Administration (NMPA).

[0118] The time-consuming laboratory testing and diagnostic difficulties of bloodstream infection pathogens (BSIs) are currently a major challenge urgently needed to be addressed by clinical emergency and infectious disease departments, posing a significant threat to human health and life. Therefore, a highly sensitive and rapid method for detecting bloodstream infection pathogens would help quickly identify the cause, reduce mortality, and effectively improve the timeliness of diagnosis and treatment, thereby enhancing medical standards and efficiency.

[0119] The rapid multiplex digital PCR (dPCR) detection method achieves absolute quantification of nucleic acid molecules by randomly partitioning a fluorescent quantitative reaction system into tens of thousands of independent, tiny reaction units based on a Poisson distribution and the proportion of positive droplets. As a third-generation PCR technology, dPCR boasts higher sensitivity than fluorescent quantitative PCR and is highly resistant to interference, enabling absolute quantification. It is suitable for challenging tests such as BSI, which involves low pathogen loads, complex blood components, and high human background DNA signals. This innovative rapid bloodstream infection pathogen detection technology based on multiplex digital PCR has a detection limit as low as 5-10 copies / reaction and shortens the reporting cycle from the current 2-3 days required for blood culture to 4 hours. This will effectively address the current global challenges of low detection rates and long reporting times for bloodstream infection pathogens.

[0120] Escherichia coli, Klebsiella pneumoniae, coagulase-negative staphylococci, and Staphylococcus aureus

[0121] Escherichia coli is a common pathogen in bloodstream infections, especially those secondary to urinary tract infections. The endotoxins it produces can lead to sepsis and septic shock.

[0122] Klebsiella pneumoniae is an important pathogen of hospital-acquired bloodstream infections, especially multidrug-resistant strains (such as carbapenemase-producing Klebsiella pneumoniae), which can cause severe sepsis and high mortality.

[0123] Coagulase-negative staphylococci (CoNS) are the primary pathogens of catheter-related bloodstream infections, particularly in immunocompromised patients or those with chronic catheter use. Although relatively low in virulence, they readily form biofilms and are difficult to eradicate. These include Staphylococcus hominis, Staphylococcus capitis, Staphylococcus simulans, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus caprae, Staphylococcus lugdunensis, Staphylococcus worderi, Staphylococcus xylosus, and Staphylococcus sciuri.

[0124] Staphylococcus aureus is the main pathogen of bloodstream infection, especially methicillin-resistant Staphylococcus aureus (MRSA), which can cause serious complications such as sepsis and endocarditis, with a high mortality rate.

[0125] In summary, coagulase-negative Staphylococci and Staphylococcus aureus are Gram-positive bacteria. The former is often seen in catheter-related infections, while the latter is associated with serious bloodstream infections, especially MRSA. Escherichia coli and Klebsiella pneumoniae are Gram-negative bacteria that are common in hospital-acquired bloodstream infections and can easily lead to sepsis.

[0126] Detection method of the present invention

[0127] The present invention provides a method for detecting pathogens that cause bloodstream infection, comprising the following steps:

[0128] 1) Design specific primers and probes (including internal reference primers and probes) based on the pathogen's genetic sequence. Pathogens include Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococci, and Staphylococcus aureus. Designing degenerate primers and probes specifically for coagulase-negative Staphylococci allows for more comprehensive detection and avoids missed detections. Specific primer and probe sequences are shown in Table 1.

[0129] Table 1

[0130]

[0131] Note: W represents A / T degenerate bases, and Y represents C / T degenerate bases.

[0132] The 5' end of the probe sequence is further connected to a fluorescent reporter gene, and the 3' end is further connected to a fluorescent quencher group. The fluorescent reporter group is selected from the group consisting of FAM, HEX, ROX, CY5, and Q705; and the fluorescent quencher group is selected from the group consisting of BHQ1, BHQ2, and BHQ3.

[0133] The above primers and probes were used for digital PCR detection, and the optimal detection conditions of the PCR system were optimized.

[0134] 2) Extract nucleic acids from the sample. Select a method appropriate for the sample type, including blood culture, peripheral blood, or peripheral blood plasma / serum. Specifically, for cell-free nucleic acid extraction from blood samples, the steps are as follows:

[0135] First, the choice of blood collection tubes. If ordinary blood collection tubes are used, they must not contain heparin and the plasma must be separated within 4 hours. If the blood cannot be processed in time, blood collection tubes with blood cell stabilization technology must be selected.

[0136] Second, separation of plasma: The first step is to remove cells by low-speed centrifugation at 4°C, 1600g, and 10 minutes.

[0137] Third, extraction: Use a plasma free nucleic acid extraction kit and follow the instructions for extraction.

[0138] 3) Digital PCR.

[0139] First, configure the PCR system. In the reagent preparation area, configure the PCR system as follows:

[0140] 5× HS Taq buffer, containing Mg 2+ 6μL, dNTPs (10mM each) 0.75μL, hot start Taq DNA polymerase 0.2μL, SEQ ID NO: 1 (10μM) 1μL, SEQ ID NO: 2 (10μM) 1μL, SEQ ID NO: 3 (10μM) 0.5μL, SEQ ID NO: 4 (10μM) 1μL, SEQ ID NO: 5 (10 μM) 1 μL, SEQ ID NO: 6 (10 μM) 0.5 μL, SEQ ID NO: 7 (10 μM) 1 μL, SEQ ID NO: 8 (10 μM) 1 μL, SEQ ID NO: 9 (10 μM) 0.5 μL, SEQ ID NO: 10 (10 μM) 1 μL, SEQ ID NO: 11(10μM) 1μL, SEQ ID NO: 12(10μM) 0.5μL, template 5μL, add water to make up to 30μL.

[0141] Preferably, the final concentration of the primers is 0.33 μM each, and the final concentration of the probes is 0.17 μM each.

[0142] Second, add template. Add template to the sample preparation area in the following order: test sample, blank control, negative control, and positive control. The negative control is normal human leukocyte genomic DNA (tgDNA) at a copy number concentration of 1000 copies / μL. The positive control solution has a copy number of 50 copies / μL for all target sites. The blank control is ultrapure water.

[0143] Third, generate droplets. Generate droplets according to the instrument requirements.

[0144] Fourth, PCR. Annealing temperature optimization experiments revealed that annealing at 56°C for 15 seconds yielded the best results for digital PCR. PCR was performed using the following protocol: 95°C for 10 minutes, 40 cycles of 95°C for 30 seconds, 56°C for 15 seconds, and 72°C for 15 seconds, with a ramp rate of 2°C / s.

[0145] Fifth, scan. Based on the scan results, determine whether the sample is positive or negative.

[0146] The test results are determined as follows:

[0147] (1) If there are ≥3 positive droplets and the cluster is consistent with the positive control, it is considered a positive result and the infectious pathogen and copy number are directly reported;

[0148] (2) If there are no positive droplets and the clusters are consistent with the negative control, it indicates a negative result and is reported as not detected;

[0149] (3) If there are 1 or 2 positive droplets, it is indicated as a gray area and retesting is recommended; if the retest still shows less than 3 positive droplets, it is determined to be a negative result and reported as not detected.

[0150] Kit of the present invention

[0151] The present invention provides a kit for detecting Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococci, and Staphylococcus aureus. The kit includes PCR primers and probes (including internal reference primers and probes), a reaction system, reaction conditions, a negative control DNA sample, and a positive control DNA sample. The following contents are provided:

[0152] Primers and probes: Synthesize the oligonucleotide fragments and modified oligonucleotide fragments shown in Table 1 and dissolve them in TE (10 μM). For details, see the previous section.

[0153] The reaction conditions are detailed in the previous section.

[0154] PCR reagents: DNA polymerase using 5× HS Taq Buffer with Mg 2+ Reaction system, see the previous section.

[0155] Controls: The blank control consisted of water. The negative control consisted of normal human leukocyte genome (tgDNA) at a copy number concentration of 1000 copies / μL. The positive control consisted of a mixture of Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococcus, and Staphylococcus aureus genomes. The copy number of all target loci in the positive control solution was 50 copies / μL.

[0156] The main advantages of the present invention include:

[0157] (a) Compared with existing technologies such as blood culture, MALD-TOF MS, and NGS, the present invention uses Taqman probes in combination with digital PCR to address problems such as low sensitivity, poor specificity, high requirements for sample type and quality, and complex positive interpretation methods.

[0158] (b) High Sensitivity: Because this method utilizes a digital PCR platform, it can divide the reaction system into approximately 20,000 micro-reactions, theoretically capable of detecting single-copy mutations. This offers a sensitivity advantage unmatched by other technologies. The detection method of this invention has been validated to achieve a minimum detection limit of 5 copies / reaction.

[0159] (c) High specificity: Specific primers and probes are designed for the specific sequences of Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococcus, and Staphylococcus aureus, respectively. They effectively amplify the target position without amplifying other sequences.

[0160] (d) It has relaxed requirements for sample type and quality, and exhibits strong resistance to interference. Due to the high sensitivity of the present invention, it is suitable for peripheral blood samples (which are relatively easy to obtain, but have low DNA content and are often fragmented). Furthermore, due to the unique nature of its digital PCR platform, it can divide the reaction system into approximately 20,000 small systems, and thus also separate interfering substances into approximately 20,000 parts. This significantly reduces the impact of interfering substances on the reaction, and of course, allows for the detection of samples with more complex backgrounds. This is something that other platforms cannot achieve.

[0161] (e) Simple positive interpretation method: Since the present invention uses an absolute quantitative method, there is no need to set up a control standard curve. The results can be determined based on the two-dimensional fluorescence graph to determine whether the target mutant template is present. The interpretation method is as follows:

[0162] (1) If there are ≥3 positive droplets and the cluster is consistent with the positive control, it is considered a positive result and the infectious pathogen and copy number are directly reported;

[0163] (2) If there are no positive droplets and the clusters are consistent with the negative control, it indicates a negative result and is reported as not detected;

[0164] (3) If there are 1 or 2 positive droplets, it is indicated as a gray area and retesting is recommended; if the retest still shows less than 3 positive droplets, it is determined to be a negative result and reported as not detected.

[0165] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are molar percentages and molar parts.

[0166] Example 1 Screening of primers and probes for detecting Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococcus, and Staphylococcus aureus by digital PCR

[0167] 1.1 Primer and probe design

[0168] Primer and probe design for pathogen detection: Multiple sets of primers and probes were designed based on the specific genes of Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococci, and Staphylococcus aureus. The sequences that exhibited no mutual interference and excellent amplification efficiency were screened and are shown in Table 2 below. The probe sequences were linked to a fluorescent reporter gene at the 5' end and a fluorescence quencher at the 3' end.

[0169] The upstream and downstream primers and probes for detecting coagulase-negative Staphylococci are designed to target a specific conserved region of the sodA gene of coagulase-negative Staphylococci. The upstream and downstream primers and probes for detecting Escherichia coli are designed to target a specific conserved region of the ydhQ gene of E. coli. The upstream and downstream primers and probes for detecting Klebsiella pneumoniae are designed to target a specific conserved region of the phoE gene of Klebsiella pneumoniae. The upstream and downstream primers and probes for detecting Staphylococcus aureus are designed to target a specific conserved region of the BioY gene of S. aureus.

[0170] Internal reference primer and probe design: Primer and probe selection was performed from multiple human genes, aiming for optimal amplification efficiency without interaction with the pathogen detection primer and probe described above. The internal reference sequences shown in Table 2 were ultimately selected and used as the internal reference sequences for the kit. The upstream and downstream primers and probes used for internal reference detection were designed to target specific conserved regions of the human EGFR gene.

[0171] Table 2

[0172]

[0173] Note: W represents A / T degenerate bases, and Y represents C / T degenerate bases.

[0174] 1.2 PCR system

[0175] 5× HS Taq buffer, containing Mg 2+ 6μL, dNTPs (10mM each) 0.75μL, hot start Taq DNA polymerase 0.2μL, SEQ ID NO: 1 (10μM) 1μL, SEQ ID NO: 2 (10μM) 1μL, SEQ ID NO: 3 (10μM) 0.5μL, SEQ ID NO: 4 (10μM) 1μL, SEQ ID NO: 5 (10 μM) 1 μL, SEQ ID NO: 6 (10 μM) 0.5 μL, SEQ ID NO: 7 (10 μM) 1 μL, SEQ ID NO: 8 (10 μM) 1 μL, SEQ ID NO: 9 (10 μM) 0.5 μL, SEQ ID NO: 10 (10 μM) 1 μL, SEQ ID NO: 11(10μM) 1μL, SEQ ID NO: 12(10μM) 0.5μL, template 5μL, add water to make up to 30μL.

[0176] 1.4 Chip Scanning

[0177] After testing, it can effectively detect Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococcus, Staphylococcus aureus and internal reference ( Figure 1-Figure 5 ). In addition, 9 types of coagulase-negative Staphylococci commonly found in clinical samples were tested, and the test results are shown in Table 3 below:

[0178] Table 3

[0179]

[0180] Comparison of the singleplex and multiplex results for each target revealed no difference in amplification concentration between the singleplex and multiplex assays (Table 4). Therefore, there was no interaction between the primers and probes that could reduce PCR amplification efficiency. Figure 6-Figure 13 .

[0181] Table 4

[0182]

[0183] Example 2: Sensitivity Verification of Digital PCR for Detecting Escherichia coli, Klebsiella pneumoniae, Coagulase-negative Staphylococci, and Staphylococcus aureus

[0184] 2.1 Experimental Methods

[0185] The strains tested include: Escherichia coli, Klebsiella pneumoniae, coagulase-negative staphylococci, Staphylococcus aureus. The coagulase-negative staphylococci include: Staphylococcus hominis, Staphylococcus capitis, Staphylococcus simulans, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus caprae, Staphylococcus lugdunensis, Staphylococcus worderi, Staphylococcus xylosus, and Staphylococcus sciuri.

[0186] The copy number (copies / μL) of each pathogen template was calculated according to Example 1, and each pathogen template was diluted to 2 copies / μL and 1 copy / μL using TE to prepare a gradient dilution template.

[0187] Verification of digital PCR system: 5× HS Taq buffer, containing Mg 2+ 6μL, dNTPs (10mM each) 0.75μL, hot start Taq DNA polymerase 0.2μL, SEQ ID NO: 1 (10μM) 1μL, SEQ ID NO: 2 (10μM) 1μL, SEQ ID NO: 3 (10μM) 0.5μL, SEQ ID NO: 4 (10μM) 1μL, SEQ ID NO: 5 (10 μM) 1 μL, SEQ ID NO: 6 (10 μM) 0.5 μL, SEQ ID NO: 7 (10 μM) 1 μL, SEQ ID NO: 8 (10 μM) 1 μL, SEQ ID NO: 9 (10 μM) 0.5 μL, SEQ ID NO: 10 (10 μM) 1 μL, SEQ ID NO: 11(10μM) 1μL, SEQ ID NO: 12(10μM) 0.5μL, template 5μL, add water to make up to 30μL.

[0188] Add serially diluted templates to the sample preparation area in the following order: 2 copies / μL, 1 copy / μL of each pathogen genome.

[0189] The PCR reaction system was generated into droplets using the same method as in Example 1. PCR was performed according to the following protocol: 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s). The plate was read according to the instrument's instructions.

[0190] 2.2 Experimental Results

[0191] Digital PCR results are shown in Figures 14-27 In addition, the digital PCR method of the present invention can effectively detect positive spots when detecting 1 cps / μL template, that is, the minimum detection sensitivity of the digital PCR method of the present invention is 5 copies / reaction.

[0192] Example 3 Specificity Verification of Digital PCR for Detection of Escherichia coli, Klebsiella pneumoniae, Coagulase-negative Staphylococci, and Staphylococcus aureus

[0193] 3.1 Experimental Methods

[0194] Design primers and probes for different pathogens. Calculate the copy number (copies / μL) of each pathogen template according to Example 1. Dilute each pathogen template to 10,000 copies / μL with TE. Then, mix the pathogens in equal proportions in groups of five (a total of eight groups or eight samples). The combinations are shown in Table 5 below:

[0195] Table 5

[0196]

[0197] Verification of digital PCR system: 5× HS Taq buffer, containing Mg 2+ 6μL, dNTPs (10mM each) 0.75μL, hot start Taq DNA polymerase 0.2μL, SEQ ID NO: 1 (10μM) 1μL, SEQ ID NO: 2 (10μM) 1μL, SEQ ID NO: 3 (10μM) 0.5μL, SEQ ID NO: 4 (10μM) 1μL, SEQ ID NO: 5 (10 μM) 1 μL, SEQ ID NO: 6 (10 μM) 0.5 μL, SEQ ID NO: 7 (10 μM) 1 μL, SEQ ID NO: 8 (10 μM) 1 μL, SEQ ID NO: 9 (10 μM) 0.5 μL, SEQ ID NO: 10 (10 μM) 1 μL, SEQ ID NO: 11(10μM) 1μL, SEQ ID NO: 12(10μM) 0.5μL, template 5μL, add water to make up to 30μL.

[0198] Add templates to the sample preparation area in the following order: blank control, negative control, and mixed pathogen template. The blank control is water, the negative control is tgDNA, and the mixed pathogen template is as shown in Table 5 above.

[0199] The PCR reaction system was generated into droplets using the same method as in Example 1. PCR was performed according to the following protocol: 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s). The plate was read according to the instrument's instructions.

[0200] 3.2 Experimental Results

[0201] Digital PCR results are shown in Figure 28 The digital PCR method of the present invention uses water or tgDNA as a template, resulting in a clean, contamination-free background. Furthermore, the digital PCR method of the present invention showed no cross-reactions when detecting common clinical pathogens other than the target at a concentration of 2000 copies / μL, demonstrating good specificity.

[0202] discuss

[0203] The present invention provides a method and application for detecting bloodstream infection pathogens, including primers and probes. More specifically, the method involves detecting pathogens including coagulase-negative Staphylococcus, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus.

[0204] The primer-probe combination provided by the present invention primarily utilizes a multiplex fluorescence PCR analysis method, designing primer probes on specific genes of different pathogens to detect and differentiate different pathogens. The Escherichia coli probe sequence has a FAM fluorescent group at its 5' end and a BHQ1 group at its 3' end; the Klebsiella pneumoniae probe sequence has a HEX fluorescent group at its 5' end and a BHQ1 group at its 3' end; the coagulase-negative Staphylococcus probe sequence has a ROX fluorescent group at its 5' end and a BHQ2 group at its 3' end; and the Staphylococcus aureus probe sequence has a CY5 fluorescent group at its 5' end and a BHQ2 group at its 3' end.

[0205] The digital PCR detection kit for blood pathogens of the present invention can detect nucleic acid fragments of pathogens in the blood, making up for the shortcomings of conventional blood culture detection methods such as low throughput and long time consumption. It provides comprehensive, accurate, and low-cost etiological diagnosis for the clinic in the first time, and provides an important reference for personalized medication and precision medicine.

[0206] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A primer-probe combination for detecting bloodstream infection pathogens, characterized in that: The primer-probe combination includes: (a) upstream and downstream primers and probes for detecting coagulase-negative Staphylococci as shown in SEQ ID NOs: 7 to 9; (b) upstream and downstream primers and probes for detecting Escherichia coli as shown in SEQ ID NOs: 1 to 3; (c) upstream and downstream primers and probes for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 4 to 6; and (d) Upstream and downstream primers and probes for detecting Staphylococcus aureus as shown in SEQ ID NOs: 10-12.

2. The primer-probe combination according to claim 1, wherein The coagulase-negative staphylococci include: Staphylococcus hominis, Staphylococcus capitis, Staphylococcus mimicus, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus caprae, Staphylococcus lugdunensis, Staphylococcus worderi, Staphylococcus xylosus and Staphylococcus squirri.

3. The primer-probe combination according to claim 1, wherein The primer-probe combination further includes: (e) upstream and downstream primers and probes for detecting internal reference genes as shown in SEQ ID NOs: 13 to 15.

4. The primer-probe combination according to claim 1, wherein The fluorescent reporter group of the coagulase-negative staphylococcus probe is ROX; the fluorescent reporter group of the Escherichia coli probe is FAM; the fluorescent reporter group of the Klebsiella pneumoniae probe is HEX; and the fluorescent reporter group of the Staphylococcus aureus probe is CY5.

5. The use of the primer-probe combination according to claim 1, wherein A kit for preparing a bloodstream infection detection kit; wherein the pathogens include: coagulase-negative staphylococci, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus.

6. A kit for detecting bloodstream infection pathogens, characterized in that: The kit comprises the primer-probe combination according to claim 1.

7. The kit according to claim 6, wherein The kit also includes a negative quality control product and a positive quality control product.

8. The kit according to claim 7, wherein The positive quality control product is a mixture of coagulase-negative Staphylococcus, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus genomes; the negative quality control product is normal human leukocyte genomic DNA.

9. The use of the primer-probe combination according to claim 1, wherein Used for preparing reagents for detecting bloodstream infection pathogens; wherein the bloodstream infection pathogens include: coagulase-negative staphylococci, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus.

10. The use according to claim 9, characterized in that The detection comprises the following steps: (i) Extracting free DNA from the sample to be tested; (ii) performing digital PCR on the free DNA obtained in step (i) using the primer-probe combination of claim 1; (iii) Read the fluorescence data and calculate the copy concentration of the target sequence to obtain the detection result.

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