A method and kit for detecting blood stream infection pathogens and application

Abstract

The present application provides a method and a kit for detecting blood stream infection pathogens and application. Specifically, the present application provides a primer probe combination for detecting blood stream infection pathogens, which comprises: (a) upper and lower primers and a probe for detecting Serratia marcescens as shown in SEQ ID NO: 7-9; (b) upper and lower primers and a probe for detecting Burkholderia cenocepacia as shown in SEQ ID NO: 10-12; (c) upper and lower primers and a probe for detecting Stenotrophomonas maltophilia as shown in SEQ ID NO: 1-3; and (d) upper and lower primers and a probe for detecting Enterobacter cloacae as shown in SEQ ID NO: 4-6. The primer probe combination of the present application can be used to achieve rapid, high sensitivity and high specificity detection of blood stream infection pathogens.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics, and more specifically, to a method, kit, and application for detecting bloodstream infectious pathogens. Background Technology

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

[0003] The incidence and mortality rates of bloodstream infections are rising annually due to factors such as increased invasive procedures and the increased use of broad-spectrum antibiotics and corticosteroids. Bloodstream infections often result in poor prognosis, prolonged hospital stays, and increased financial burden on patients. Early identification of the causative agent and selection of appropriate antibiotics can reduce mortality by approximately 7%. Therefore, timely and rapid identification of the causative agent of bloodstream infections is crucial.

[0004] For a long time, there have been many problems in the clinical diagnosis and treatment of BSI, such as the need for continuous improvement in early rapid diagnosis and precise treatment. At present, the main detection methods for BSI are: (1) blood culture, but it has shortcomings such as low detection rate, long detection time, and high contamination and false positive rate; (2) MALD-TOF MS technology, which has good identification efficacy for Gram-negative bacteria (>90% consistent with subsequent culture results), but it still needs to be used with caution when identifying Gram-positive bacteria (about 80% consistent with subsequent culture results); (3) next-generation sequencing (NGS) technology, which has high detection cost and is therefore not suitable for rapid detection required by general bloodstream infection patients. In addition, next-generation sequencing requires the DNA of the sample to be tested to be enriched by PCR first, so there will be amplification bias for some segments, which will lead to missed detection and insufficient accuracy.

[0005] Therefore, there is a need to develop a rapid, highly specific, highly sensitive, and interference-resistant method and kit for detecting bloodstream infections. Summary of the Invention

[0006] This invention provides a rapid, highly specific, highly sensitive, and interference-resistant method and kit for detecting bloodstream infections.

[0007] In a first aspect of the invention, a primer-probe combination (or primer-probe composition) for detecting bloodstream pathogens is provided, said primer-probe combination (or primer-probe composition) being selected from the group consisting of:

[0008] (a) Upstream and downstream primers and probes for detecting Serratia marcescens, as shown in SEQ ID NO:7-9.

[0009] (b) Upstream and downstream primers and probes for detecting Burkholderia cepacia, as shown in SEQ ID NO:10-12;

[0010] (c) Upstream and downstream primers and probes for detecting Stenotrophomonas maltophilia, as shown in SEQ ID NO:1-3;

[0011] (d) Upstream and downstream primers and probes for detecting Enterobacter cloacae, as shown in SEQ ID NO:4-6;

[0012] (f) Any combination of two or more of (a) to (d) above.

[0013] In a preferred aspect of the invention, a primer-probe combination (or primer-probe composition) for detecting bloodstream pathogens is provided, the primer-probe combination (or primer-probe composition) comprising:

[0014] (a) Upstream and downstream primers and probes for detecting *Serratia marcescens* as shown in SEQ ID NO: 7–9. In another preferred embodiment, the primer-probe combination further includes primer-probe combinations selected from the group consisting of:

[0015] (b) Upstream and downstream primers and probes for detecting Burkholderia cepacia, as shown in SEQ ID NO:10-12;

[0016] (c) Upstream and downstream primers and probes for detecting Stenotrophomonas maltophilia, as shown in SEQ ID NO:1-3;

[0017] (d) Upstream and downstream primers and probes for detecting Enterobacter cloacae, as shown in SEQ ID NO:4-6.

[0018] (e) At least two of the primer-probe combinations mentioned in (b) to (d) above.

[0019] In a preferred aspect of the present invention, a primer-probe combination for detecting bloodstream pathogens is provided, the primer-probe combination comprising:

[0020] (a) Upstream and downstream primers and probes for detecting Serratia marcescens, as shown in SEQ ID NO:7-9;

[0021] (b) Upstream and downstream primers and probes for detecting Burkholderia cepacia, as shown in SEQ ID NO:10-12;

[0022] (c) The upstream and downstream primers and probes for detecting Stenotrophomonas maltophilia, as shown in SEQ ID NO: 1–3; and

[0023] (d) Upstream and downstream primers and probes for detecting Enterobacter cloacae, as shown in SEQ ID NO:4-6.

[0024] In another preferred embodiment, the primer-probe combination further includes primers and probes for detecting other bloodstream pathogens.

[0025] In another preferred embodiment, the primer-probe combination and primer-probe composition have the same meaning and can be used interchangeably.

[0026] In another preferred embodiment, the primer-probe combination further includes:

[0027] (e) Upstream and downstream primers and probes for detecting internal reference (or internal reference gene) as shown in SEQ ID NO:17-19.

[0028] In another preferred embodiment, the primers and probes used for detecting the internal reference are designed for a specific conserved region of the human EGFR gene.

[0029] In this article, "dissimilar and non-interfering" means that each probe in the composition uses a different fluorophore and will not affect the detection of each other, i.e., different channels can be used for detection. For example, FAM, HEX, ROX, CY5 and Q705 can be used. These groups have different absorbance values ​​and can be selected in different channels, so they will not interfere with each other.

[0030] In another preferred embodiment, the fluorescent reporter group of the *Serratia marcescens* probe is ROX; the fluorescent reporter group of the *Burkholderia cepacia* probe is CY5; the fluorescent reporter group of the *Stenotrophomonas maltophilia* probe is FAM; and the fluorescent reporter group of the *Enterobacter cloacae* probe is HEX.

[0031] In another preferred embodiment, the fluorescent reporter group of the internal reference probe is Q705.

[0032] In another preferred embodiment, the 3' end of the probe also has a fluorescence quenching group.

[0033] In another preferred embodiment, the fluorescence quenching group is selected from the group consisting of BHQ1, BHQ2, and BHQ3.

[0034] In another preferred embodiment, the fluorescence quenching group at the 3' end of the Stenotrophomonas maltophilia probe and the Enterobacter cloacae probe is BHQ1; and the fluorescence quenching group at the 3' end of the Serratia marcescens probe and the Burkholderia cepacia probe is BHQ2.

[0035] In another preferred embodiment, the fluorescence quenching group at the end of the internal reference probe is BHQ3.

[0036] In another preferred embodiment, each component of the primer-probe combination is contained in a separate package.

[0037] In another preferred embodiment, the components of the primer-probe combination are contained in the same package.

[0038] In another preferred embodiment, the components of the combination exist in a mixed form.

[0039] In another preferred embodiment, the probe sequence has a fluorescent reporter gene linked to its 5' end and a fluorescent quencher linked to its 3' end.

[0040] In a second aspect of the invention, the use of the primer-probe combination as described in the first aspect of the invention is provided for preparing a kit for detecting bloodstream infection pathogens; wherein the pathogens include: Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

[0041] In a third aspect of the invention, a kit for detecting bloodstream pathogens is provided, the kit comprising the primer-probe combination as described in the first aspect of the invention.

[0042] In another preferred embodiment, the kit further includes negative and positive controls.

[0043] In another preferred embodiment, the negative control is at least one of DEPC-H2O, physiological saline, internal standard gene pseudovirus, and normal human leukocyte genomic DNA.

[0044] In another preferred embodiment, the negative control is genomic DNA from normal human leukocytes.

[0045] In another preferred embodiment, the copy number concentration of the genomic DNA of the normal human leukocytes is 1000 copies / μL.

[0046] In another preferred embodiment, the kit further includes:

[0047] (e) Upstream and downstream primers and probes for detecting internal controls, as shown in SEQ ID NO:17-19.

[0048] In another preferred embodiment, the positive control is at least one of the following: Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

[0049] In another preferred embodiment, the positive control is a mixture of the genomes of Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

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

[0051] In another preferred embodiment, the kit further includes a PCR reaction solution.

[0052] In another preferred embodiment, the PCR reaction solution includes dNTPs, PCR buffer, and Mg. 2+ And at least one of DNA polymerases.

[0053] In another preferred embodiment, the DNA polymerase is 5×HS Taq Buffer with Mg 2+ Reaction system.

[0054] In another preferred embodiment, the kit also includes an instruction manual stating that the kit is used to detect bloodstream pathogens.

[0055] In another preferred embodiment, the kit further includes a blank control, which is ultrapure water.

[0056] In a fourth aspect of the invention, the use of the primer-probe combination as described in the first aspect of the invention or the kit as described in the third aspect of the invention is provided for the preparation of reagents for detecting bloodstream pathogens.

[0057] In another preferred embodiment, the detection includes the following steps:

[0058] (i) Extracting cell-free DNA from the sample to be tested;

[0059] (ii) Perform quantitative real-time 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;

[0060] (iii) Read the fluorescence data and calculate the copy concentration of the target sequence to obtain the detection results.

[0061] In another preferred embodiment, in step (iii), the determination of the detection result is as follows:

[0062] (1) If there are ≥3 positive droplets and the clustering is consistent with the positive control, it indicates a positive result, and the infecting pathogen and copy number should be reported directly;

[0063] (2) If there are no positive droplets and the clustering is consistent with the negative control, it indicates a negative result and the report indicates no detection.

[0064] (3) If there are 1 or 2 positive droplets, it indicates a gray area and a retest is recommended; if the retest still shows <3 positive droplets, it is considered a negative result and the report indicates no detection.

[0065] In another preferred embodiment, the sample to be tested is selected from the group consisting of whole blood, plasma, serum, or combinations thereof.

[0066] In another preferred embodiment, the bloodstream infection pathogens include: Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

[0067] In a fifth aspect of the invention, a method for detecting bloodstream infection pathogens is provided, comprising the following steps:

[0068] (i) Extracting cell-free DNA from the sample to be tested;

[0069] (ii) Perform quantitative real-time 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;

[0070] (iii) Read the fluorescence data and calculate the copy concentration of the target sequence to obtain the detection results.

[0071] In another preferred embodiment, the bloodstream infection pathogens include: Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

[0072] In another preferred embodiment, in step (iii), the determination of the detection result is as follows:

[0073] (1) If there are ≥3 positive droplets and the clustering is consistent with the positive control, it indicates a positive result, and the infecting pathogen and copy number should be reported directly;

[0074] (2) If there are no positive droplets and the clustering is consistent with the negative control, it indicates a negative result and the report indicates no detection.

[0075] (3) If there are 1 or 2 positive droplets, it indicates a gray area and a retest is recommended; if the retest still shows <3 positive droplets, it is considered a negative result and the report indicates no detection.

[0076] In another preferred embodiment, the sample to be tested is selected from the group consisting of whole blood, plasma, serum, or combinations thereof.

[0077] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0078] In another preferred embodiment, the method is an in vitro method.

[0079] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described 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 described in detail here. Attached Figure Description

[0080] Figure 1 The image shows the detection results of Stenotrophomonas maltophilia detected by multiplex digital PCR using the primer-probe combination of the present invention.

[0081] Figure 2 The image shows the detection results of Enterobacter cloacae detected by multiplex digital PCR using the primer-probe combination of the present invention.

[0082] Figure 3 The graph shows the detection results of Serratia marcescens using multiplex digital PCR with the primer-probe combination of the present invention.

[0083] Figure 4 The image shows the detection results of Burkholderia cepacia detected by multiplex digital PCR using the primer-probe combination of the present invention.

[0084] Figure 5 The graph shows the detection results of the internal reference detected by multiplex digital PCR using the primer-probe combination of the present invention.

[0085] Figure 6 The detection sensitivity of Stenotrophomonas maltophilia in Example 2 is shown.

[0086] Figure 7 The detection sensitivity of Enterobacter cloacae in Example 2 is shown.

[0087] Figure 8 The detection sensitivity of Serratia marcescens in Example 2 is shown.

[0088] Figure 9 The detection sensitivity of Burkholderia cepacia in Example 2 is shown.

[0089] General description of the attached figures: The gray markings in the attached figures indicate supplementary explanations of the figures, used to indicate the specific single-channel fluorescence information of the individual figures below, that is, the signal intensity measured by the droplet in a specific fluorescence detection channel (such as FAM, HEX, ROX, CY5). Detailed Implementation

[0090] Through extensive and in-depth research and screening, the inventors unexpectedly developed primers and probes for the rapid, highly specific, and sensitive detection of bloodstream infection pathogens. These pathogens include *Serratia marcescens*, *Burkholderia cepacia*, *Stenotrophomonas maltophilia*, and *Enterobacter cloacae*. Experiments show that the digital PCR method of this invention has a minimum detection sensitivity of 5 copies / reaction and good specificity. Based on these findings, this invention was completed.

[0091] the term

[0092] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0093] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0094] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0095] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0096] Bloodstream infection

[0097] Bloodstream infection (BSI) refers to infection caused by pathogenic microorganisms entering the bloodstream. The main pathogens include bacteria, fungi, and viruses. BSI is characterized by three highs and one difficulty: high morbidity, high mortality, high treatment costs, and difficulty in diagnosis.

[0098] BSI has a 40% chance of developing into sepsis. Studies have found that for every hour of delay in treatment after the onset of hypotension in septic shock patients, the survival rate decreases by 7.6%, with a median survival rate of 42% after a 6-hour delay.

[0099] Currently, blood culture is the gold standard for BSI laboratory diagnosis, but its positive rate is only around 10%, and it takes at least 2-3 days to report the results. There are also some molecular detection methods, such as bioMérieux's FilmArray BCID, Luminex's Verigene Gram+BC and Verigene Gram-BC, but these methods are all based on positive blood cultures. Some mNGS-based detection methods are also available in China, but mNGS testing takes 1-2 days, is costly, complex, and requires specialized bioinformatics analysts, making it difficult to widely implement in medical institutions.

[0100] Bloodstream infection (BSI) pathogen laboratory testing is time-consuming and difficult to diagnose, posing a significant challenge to clinical emergency departments and infectious disease departments that urgently need to be addressed and pose a serious threat to public health. Therefore, a highly sensitive and rapid detection method for bloodstream infection pathogens will help quickly identify the cause, reduce mortality, effectively improve the timeliness of diagnosis and treatment, enhance medical standards, and increase medical efficiency.

[0101] Multiplex digital PCR (dPCR) is a rapid detection method that randomly divides the quantitative PCR reaction system into tens of thousands of independent micro-reaction units and achieves absolute quantification of nucleic acid molecules based on Poisson distribution and the proportion of positive droplets. As a third-generation PCR technology, dPCR has higher sensitivity than quantitative PCR, stronger anti-interference capabilities, and can achieve absolute quantification. It is suitable for challenging detection of bloodstream infection pathogens such as BSI, which has low pathogen load, complex blood composition, and high human background DNA signal. 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 the reporting cycle is shortened from the current 2-3 days for blood culture to 4 hours, effectively solving the global challenges of low detection rates and long reporting cycles for bloodstream infection pathogens.

[0102] Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae

[0103] Serratia marcescens is a Gram-negative bacillus belonging to the Enterobacteriaceae family. It can enter the bloodstream through medical devices (such as catheters) or surgical wounds, causing bloodstream infections (bacteremia). It is common in immunocompromised patients, especially those who are hospitalized for extended periods or undergo invasive treatments. Infection can lead to sepsis, which can be life-threatening in severe cases.

[0104] Burkholderia cepacia is a Gram-negative bacillus widely found in soil and water. It can enter the bloodstream through respiratory infections or medical devices (such as catheters), causing bloodstream infections. It is naturally resistant to many antibiotics, making treatment difficult and potentially leading to sepsis or multiple organ failure.

[0105] Stenotrophomonas maltophilia is a Gram-negative bacillus widely found in water, soil, and hospital environments. It can enter the bloodstream through medical devices (such as catheters) or respiratory infections, causing bloodstream infections. It exhibits natural resistance to many antibiotics, making treatment difficult and potentially leading to sepsis or septic shock.

[0106] Enterobacter cloacae is a Gram-negative bacillus belonging to the Enterobacteriaceae family. It can enter the bloodstream through medical devices (such as catheters), surgical wounds, or intestinal translocation, causing bloodstream infections. It is resistant to many antibiotics, especially strains that produce extended-spectrum β-lactamases (ESBLs), which may lead to sepsis or septic shock.

[0107] In summary, all of the bacteria mentioned above are opportunistic pathogens, commonly found in hospital environments, especially in immunocompromised patients or those undergoing invasive treatments. Treatment of bloodstream infections requires selection of appropriate antibiotics based on pathogen detection results.

[0108] The detection method of the present invention

[0109] This invention provides a method for detecting pathogens causing bloodstream infections, including *Serratia marcescens*, *Burkholderia cepacia*, *Stenotrophomonas maltophilia*, and *Enterobacter cloacae*. The specific experimental steps are as follows:

[0110] 1) Specific primers and probes (including internal control primers and probes) were designed based on the gene sequences of the pathogens. The pathogens included *Serratia marcescens*, *Burkholderia cepacia*, *Stenotrophomonas maltophilia*, and *Enterobacter cloacae*. Digital PCR detection was performed using the aforementioned primers and probes, and the optimal detection conditions for the PCR system were optimized. This method was applied to the detection of patient blood samples. Specific primer and probe sequences are shown in Table 1.

[0111] Table 1

[0112]

[0113] The probe sequence includes a fluorescent reporter gene attached to its 5' end and a fluorescent quencher attached to its 3' end. The fluorescent reporter gene is selected from the following group: FAM, HEX, ROX, CY5, and Q705; the fluorescent quencher is selected from the following group: BHQ1, BHQ2, and BHQ3.

[0114] Digital PCR detection was performed using the aforementioned primers and probes, and the optimal detection conditions for the PCR system were optimized.

[0115] 2) Extract nucleic acid from the sample. Select a method suitable for the sample type, including peripheral blood and peripheral blood plasma / serum. Specifically, for the extraction of cell-free nucleic acid from blood samples, the steps are as follows:

[0116] First, the choice of blood collection tubes. If ordinary blood collection tubes are used, they must not contain heparin, and plasma must be separated within 4 hours. If the blood cannot be processed in time, blood collection tubes containing blood cell stabilization technology should be selected.

[0117] Second, plasma separation. Plasma separation: The first step is low-speed centrifugation to remove cells, 4℃, 1600g, 10min.

[0118] Third, extraction. Use a plasma-free nucleic acid extraction kit and follow the instructions for extraction.

[0119] 3) Digital PCR.

[0120] First, prepare the PCR system. Prepare the PCR system in the reagent preparation area according to the table below:

[0121] 5×HS Taq Buffer with Mg 2+ 6μL, dNTPs (10mM each) 0.75μL, HotStart Taq DNAPolymerase 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 (10 μM) 1 μL, SEQ ID NO:12 (10 μM) 0.5 μL, template 5 μL, add water to make up to 30 μL.

[0122] Preferably, the final concentration of each primer is 0.33 μM and the final concentration of each probe is 0.17 μM.

[0123] Second, add the template. In the sample preparation area, add the template in the following order: test sample, blank control, negative control, and positive control. The negative control is genomic DNA from normal human leukocytes at a concentration of 1000 copies / μL, and the positive control solution has a copy number of 50 copies / μL for all target sites. The blank control is ultrapure water.

[0124] Third, droplet generation. Droplet generation is performed according to the instrument requirements.

[0125] Fourth, PCR. After annealing temperature optimization experiments, it was found that annealing at 56℃ for 15s yielded the best digital PCR results. PCR was performed according to the following procedure: 95℃ for 10min, 40 cycles (95℃ for 30s, 56℃ for 15s, 72℃ for 15s), with a heating / cooling rate of 2℃ / s.

[0126] Fifth, read the plate. Based on the plate reading results, determine whether the sample is positive or negative.

[0127] The determination of the test results is as follows:

[0128] (1) If there are ≥3 positive droplets and the clustering is consistent with the positive control, it indicates a positive result, and the infecting pathogen and copy number should be reported directly;

[0129] (2) If there are no positive droplets and the clustering is consistent with the negative control, it indicates a negative result and the report indicates no detection.

[0130] (3) If there are 1 or 2 positive droplets, it indicates a gray area and a retest is recommended; if the retest still shows <3 positive droplets, it is considered a negative result and the report indicates no detection.

[0131] The reagent kit of the present invention

[0132] This invention provides a kit for detecting *Serratia marcescens*, *Burkholderia cepacia*, *Stenotrophomonas maltophilia*, and *Enterobacter cloacae*. The kit includes PCR primers and probes, a reaction system, reaction conditions, a negative control DNA sample, and a positive control DNA sample. The following content:

[0133] Primers and probes (including internal reference primers and probes): synthesize oligonucleotide fragments and modified oligonucleotide fragments as shown in Table 1, dissolved in TE (10 μM), as detailed in the preceding section.

[0134] The reaction conditions are detailed in the preceding section.

[0135] PCR reagents: DNA polymerase using 5×HS Taq Buffer with Mg 2+ The reaction system is described in the preceding section.

[0136] Controls: The blank control was water; the negative control was the genome of normal human leukocytes at a copy concentration of 1000 copies / μL. The positive control was a mixture of genomes from *Escherichia coli*, *Klebsiella pneumoniae*, coagulase-negative staphylococci, and *Staphylococcus aureus*. The copy number of all target sites in the positive control solution was 50 copies / μL.

[0137] The main advantages of this invention include:

[0138] (a) Compared with existing technologies such as blood culture, MALD-TOF MS, and NGS, this invention uses Taqman probes combined with digital PCR, which can solve problems such as low sensitivity, poor specificity, high requirements for sample type and quality, and complex positive interpretation methods.

[0139] (b) High sensitivity: Because this method uses a digital PCR platform, it can divide the reaction system into approximately 20,000 tiny reactions, theoretically capable of detecting single-copy mutations, possessing a sensitivity advantage unmatched by other technologies. The detection method of this invention has been verified to achieve a minimum detection limit of 5 copies / reaction.

[0140] (c) High specificity: The designed specific primers and probes target the specific sequences of Stenotrophomonas maltophilia, Enterobacter cloacae, Serratia marcescens, and Burkholderia cepacia, respectively, and can specifically amplify the target positions.

[0141] (d) The primer-probe combination of this invention has relaxed requirements on sample type and quality, and strong anti-interference ability. Due to the high sensitivity of this invention, it is suitable for peripheral blood samples (which are relatively easy to obtain, but have low DNA content and are fragmented); and, due to the uniqueness of its digital PCR platform, it can divide the reaction system into approximately 20,000 small systems, and at the same time, it can also divide interfering substances into approximately 20,000 portions, which can greatly reduce the influence of interfering substances on the reaction, and of course, it can detect samples with more complex backgrounds. This is something that other platforms cannot do.

[0142] (e) The positive interpretation method of this invention is simple: Since this invention uses an absolute quantitative method, there is no need to set a control standard curve. The presence of the target mutant template can be determined based on the two-dimensional fluorescence graph. The interpretation method is as follows:

[0143] (1) If there are ≥3 positive droplets and the clustering is consistent with the positive control, it indicates a positive result, and the infecting pathogen and copy number should be reported directly;

[0144] (2) If there are no positive droplets and the clustering is consistent with the negative control, it indicates a negative result and the report indicates no detection.

[0145] (3) If there are 1 or 2 positive droplets, it indicates a gray area and a retest is recommended; if the retest still shows <3 positive droplets, it is considered a negative result and the report indicates no detection.

[0146] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise 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 stated, percentages and parts are weight percentages and parts by weight.

[0147] Example 1: Detection of Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae using digital PCR.

[0148] 1.1 Primer and probe design

[0149] Primer and probe design for pathogen detection: Multiple sets of primers and probes were designed based on the specific genes of *Serratia marcescens*, *Burkholderia cepacia*, *Stenotrophomonas maltophilia*, and *Enterobacter cloacae* (Table A). The sequences that did not interfere with each other and had superior amplification efficiency were finally screened, as shown in Table 2 below. During detection, a fluorescent reporter gene was attached to the 5' end of the probe sequence, and a fluorescence quencher group was attached to the 3' end.

[0150] Primer and probe design for the internal control: Primers and probes were screened from multiple human genes to ensure no interaction with the pathogen primers and probes used for detection and to achieve optimal amplification efficiency. The resulting internal control sequences are shown in Table 2 below and are used as the internal control sequences in the kit. The upstream and downstream primers and probes used to detect the internal control were designed targeting specific conserved regions of the human EGFR gene.

[0151] Table 2

[0152]

[0153]

[0154] Table A. Specific genes and their sequence information for *Serratia marcescens*, *Burkholderia cepacia*, *Stenotrophomonas maltophilia*, and *Enterobacter cloacae*.

[0155]

[0156]

[0157]

[0158] Note: In the specific gene sequences of each pathogen in the table, the underlined part is the amplicon location, the bolded parts at both ends of the amplicon are the upstream and downstream primer locations, and the italicized and bolded parts in the amplicon are the probe locations.

[0159] 1.2 PCR system

[0160] 5×HS Taq Buffer with Mg 2+ 6μL, dNTPs (10mM each) 0.75μL, HotStart Taq DNAPolymerase 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 (10 μM) 1 μL, SEQ ID NO:12 (10 μM) 0.5 μL, template 5 μL, add water to make up to 30 μL.

[0161] 1.3 PCR program: 95℃ for 10 min, 40 cycles (95℃ for 30 s, 56℃ for 15 s, 72℃ for 15 s).

[0162] 1.4 Chip Scanning

[0163] After testing, it can effectively detect Stenotrophomonas maltophilia, Enterobacter cloacae, Serratia marcescens, and Burkholderia cepacia, such as... Figures 1-5 As shown.

[0164] Example 2: Sensitivity Verification of Digital PCR for Detection of Stenotrophomonas maltophilia, Enterobacter cloacae, Serratia marcescens, and Burkholderia cepacia

[0165] 2.1 Experimental Methods

[0166] The copy number (copies / μL) of each pathogen template was calculated according to Example 1. Each pathogen template was diluted to 2 copies / μL and 1 copies / μL using TE to prepare serially diluted templates.

[0167] Validation of digital PCR system: 5×HS Taq Buffer with Mg 2+6μL, dNTPs (10mM each) 0.75μL, HotStart 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 (10 μM) 1 μL, SEQ ID NO:12 (10 μM) 0.5 μL, template 5 μL, add water to make up to 30 μL.

[0168] In the sample preparation area, add serially diluted templates in the following order: 2 copies / μL, 1 copies / μL of each pathogen genome.

[0169] The PCR reaction system was generated into droplets using the same method as in Example 1. PCR was performed according to the PCR program: 95℃ for 10 min, 40 cycles (95℃ for 30 s, 56℃ for 15 s, 72℃ for 15 s). Plate reading was initiated according to the instrument requirements.

[0170] 2.2 Experimental Results

[0171] The results of digital PCR can be found in Figures 6-9 Furthermore, the digital PCR method of this invention can effectively detect positive spots with a template density of 1 copy / μL, meaning that the minimum detection sensitivity of the digital PCR method of this invention is 5 copies / reaction.

[0172] Example 3: Specificity Verification of Digital PCR for Detection of Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae

[0173] 3.1 Experimental Methods

[0174] Based on the type of clinical pathogen infection, the following strains were purchased, and their genomic DNA was extracted. After extraction, digital PCR detection was performed according to the method in Example 1 at a sample loading amount of 0.1 ng. The results are shown in Table 3 below.

[0175] Table 3

[0176]

[0177]

[0178] 3.2 Experimental Results

[0179] The results showed that only Stenotrophomonas maltophilia, Enterobacter cloacae, Serratia marcescens, and Burkholderia cepacia were effectively detected, while the remaining strains were undetectable. This indicates that the digital PCR method of this invention has good specificity.

[0180] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A primer-probe combination for detecting bloodstream pathogens, characterized in that, The primer-probe combination includes: (a) Upstream and downstream primers and probes for detecting Serratia marcescens, as shown in SEQ ID NO: 7-9; (b) Upstream and downstream primers and probes for detecting Burkholderia cepacia, as shown in SEQ ID NO: 10-12; (c) Upstream and downstream primers and probes for detecting Stenotrophomonas maltophilia, as shown in SEQ ID NO: 1-3; (d) Upstream and downstream primers and probes for detecting Enterobacter cloacae, as shown in SEQ ID NO: 4-6; and (e) Upstream and downstream primers and probes for detecting internal reference genes, as shown in SEQ ID NO: 17-19.

2. The primer-probe combination as described in claim 1, characterized in that, The upstream and downstream primers and probes used to detect the internal reference gene were designed for specific conserved regions of the human EGFR gene.

3. The primer-probe combination as described in claim 1, characterized in that, The fluorescent reporter group of the *Serratia marcescens* probe is ROX; the fluorescent reporter group of the *Burkholderia cepacia* probe is CY5; the fluorescent reporter group of the *Stenotrophomonas maltophilia* probe is FAM; and the fluorescent reporter group of the *Enterobacter cloacae* probe is HEX.

4. The primer-probe combination as described in claim 1, characterized in that, The fluorescent reporter group of the internal reference probe is Q705.

5. The primer-probe combination as described in claim 1, characterized in that, The probe also has a fluorescence quenching group at its 3' end.

6. The primer-probe combination as described in claim 5, characterized in that, The fluorescence quenching groups of the *Serratia marcescens* probe and the *Burkholderia cepacia* probe are both BHQ2; the fluorescence quenching groups of the *Stenotrophomonas maltophilia* probe and the *Enterobacter cloacae* probe are BHQ1.

7. The primer-probe combination as described in claim 1, characterized in that, The components of the primer-probe combination exist in a mixed form.

8. The use of the primer-probe combination as described in claim 1, characterized in that, This kit is used to prepare a reagent for detecting bloodstream infection pathogens, wherein the pathogens include: Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

9. A kit for detecting bloodstream infection pathogens, characterized in that, The kit includes the primer-probe combination as described in claim 1.

10. The kit according to claim 9, characterized in that, The kit also includes negative and positive controls.

11. The kit according to claim 10, characterized in that, The positive control is a mixture of genomes of Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae; the negative control is genomic DNA of normal human leukocytes.

12. The kit as described in claim 9, characterized in that, The kit also includes an instruction manual, which states that the kit is used to detect bloodstream infection pathogens, and that the bloodstream infection pathogens include: Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

13. The use of the primer-probe combination as described in claim 1 or the kit as described in claim 9, characterized in that, Used to prepare reagents for detecting bloodstream infection pathogens; The bloodstream infection pathogens include: Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

14. The use as described in claim 13, characterized in that, The detection includes the following steps: (i) Extracting cell-free DNA from the sample to be tested; (ii) Perform quantitative real-time PCR on the free DNA obtained in step (i) using the primer-probe combination of claim 1 or the kit of claim 9; (iii) Read the fluorescence data and calculate the copy concentration of the target sequence to obtain the detection results.

15. The use as described in claim 13, characterized in that, In step (iii), the detection result is determined as follows: (1) If there are ≥3 positive droplets and the clustering is consistent with the positive control, it indicates a positive result, and the infecting pathogen and copy number should be reported directly; (2) If there are no positive droplets and the clustering is consistent with the negative control, it indicates a negative result and the report indicates no detection. (3) If there is one or two positive droplets, it indicates a gray area and a retest is recommended; If the retest still shows <3 positive droplets, the result is considered negative and the report indicates no detection.

16. The use as described in claim 14, characterized in that, The test sample is selected from the following group: whole blood, plasma, serum, or a combination thereof.

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