Primer probe combination and kit for detecting bloodstream infection pathogens and application of primer probe combination and kit

Abstract

The invention provides a primer probe combination and a kit for detecting bloodstream infection pathogens and application of the primer probe combination and the kit. Specifically, the invention provides a primer probe combination for detecting blood flow infection pathogens, and the primer probe combination comprises: (a) upstream and downstream primers and probes for detecting pseudomonas aeruginosa as shown in SEQ ID NO: 1-3; (b) upstream and downstream primers and probes for detecting acinetobacter baumannii as shown in SEQ ID NO: 4-6; (c) an upstream primer, a downstream primer and a probe which are shown as SEQ ID NO: 7-9 and are used for detecting enterococcus; and (d) upstream and downstream primers and a probe which are shown as SEQ ID NO: 10-12 and are used for detecting streptococcus. The primer probe combination provided by the invention can be used for realizing rapid, high-sensitivity and high-specificity detection of blood flow 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] Bloodstream infection (BSI) refers to the presence of pathogenic microorganisms in a patient's blood. Pathogens that cause BSI 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 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 need to be continuously improved. The current detection methods of 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 rapid detection required by general bloodstream infection patients. In addition, NGS requires 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 Pseudomonas aeruginosa as shown in SEQ ID NOs: 1 to 3;

[0009] (b) upstream and downstream primers and probes for detecting Acinetobacter baumannii as shown in SEQ ID NOs: 4 to 6;

[0010] (c) upstream and downstream primers and probes for detecting Enterococcus as shown in SEQ ID NOs: 7 to 9; and

[0011] (d) Upstream and downstream primers and probes for detecting Streptococcus as shown in SEQ ID NOs: 10 to 12.

[0012] In another preferred embodiment, the primer-probe combination further includes primers and probes for detecting other bloodstream infection pathogens.

[0013] In another preferred embodiment, the primer-probe combination and the primer-probe composition have the same meaning and can be used interchangeably.

[0014] In another preferred embodiment, the primer-probe combination further comprises:

[0015] (e) Upstream and downstream primers and probes for detecting an internal reference (or internal reference gene) as shown in SEQ ID NOs: 13-15.

[0016] 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.

[0017] In another preferred embodiment, the Enterococcus genus includes: Enterococcus faecalis, Enterococcus faecium, Enterococcus leuproreus, Enterococcus durase and Enterococcus microenteritis.

[0018] In another preferred embodiment, the Streptococcus genus includes: Streptococcus pneumoniae, Streptococcus mitis, Streptococcus agalactiae, Streptococcus sanguis and Streptococcus salivarius.

[0019] 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.

[0020] 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.

[0021] In another preferred embodiment, the fluorescent reporter group of the Pseudomonas aeruginosa probe is FAM; the fluorescent reporter group of the Acinetobacter baumannii probe is HEX; the fluorescent reporter group of the Enterococcus probe is ROX; and the fluorescent reporter group of the Streptococcus probe is CY5.

[0022] In another preferred embodiment, the internal reference probe fluorescent reporter group is Q705.

[0023] In another preferred embodiment, the 3' end of the probe further has a fluorescence quenching group.

[0024] In another preferred embodiment, the fluorescence quenching group is selected from the following group: BHQ1, BHQ2, and BHQ3.

[0025] In another preferred embodiment, the quencher group at the 3' end of the Pseudomonas aeruginosa probe and the Acinetobacter baumannii probe is BHQ1; the quencher group at the 3' end of the Enterococcus probe and the Streptococcus probe is BHQ2.

[0026] In another preferred embodiment, the fluorescence quenching group at the end of the internal reference probe is BHQ3.

[0027] In another preferred embodiment, each component of the primer-probe combination is present in a separate package.

[0028] In another preferred embodiment, the components of the primer-probe combination are present in the same package.

[0029] In another preferred embodiment, the components of the combination are present in a mixed form.

[0030] 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.

[0031] In a second aspect of the present invention, there is provided a use of the primer-probe combination as described in the first aspect of the present invention for preparing a kit for detecting bloodstream infection pathogens; wherein the pathogens include: Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus and Streptococcus.

[0032] In another preferred embodiment, the Enterococcus genus includes: Enterococcus faecalis, Enterococcus faecium, Enterococcus leuproreus, Enterococcus durase and Enterococcus microenteritis.

[0033] In another preferred embodiment, the Streptococcus genus includes: Streptococcus pneumoniae, Streptococcus mitis, Streptococcus agalactiae, Streptococcus sanguis and Streptococcus salivarius.

[0034] 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.

[0035] In another preferred embodiment, the kit further comprises a negative control product and a positive control product.

[0036] 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.

[0037] In another preferred embodiment, the negative control product is normal human leukocyte genomic DNA (tgDNA).

[0038] In another preferred embodiment, the copy number concentration of the tgDNA is 1000 copies / μL.

[0039] In another preferred embodiment, the positive quality control product is at least one of a fragment plasmid or a fragment DNA of Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus and Streptococcus.

[0040] In another preferred embodiment, the positive quality control product is a mixture of Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus and Streptococcus genomes.

[0041] In another preferred embodiment, the copy number of all target sites in the positive quality control product is 50 copies / μL.

[0042] In another preferred embodiment, the kit further comprises:

[0043] (e) Upstream and downstream primers and probes for detecting internal controls as shown in SEQ ID NOs: 13-15.

[0044] In another preferred embodiment, the kit further comprises a PCR reaction solution.

[0045] In another preferred embodiment, the PCR reaction solution includes dNTP, PCR buffer, Mg 2+ and at least one of a DNA polymerase.

[0046] In another preferred embodiment, the DNA polymerase is 5×HS Taq Buffer with Mg 2+ reaction system.

[0047] In another preferred embodiment, the kit further comprises an instruction manual, which indicates that the kit is used to detect bloodstream infection pathogens.

[0048] In another preferred embodiment, the kit further comprises a blank control, which is ultrapure water.

[0049] 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.

[0050] In another preferred embodiment, the detection comprises the following steps:

[0051] (i) extracting free DNA from the sample to be tested;

[0052] (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;

[0053] (iii) Read the fluorescence data and calculate the copy concentration of the target sequence to obtain the detection result.

[0054] In another preferred embodiment, in step (iii), the determination of the test result is as follows:

[0055] (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;

[0056] (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;

[0057] (3) If there are 1 or 2 positive droplets, it is indicated as a gray area and re-testing is recommended; if the re-test still shows less than 3 positive droplets, it is determined to be a negative result and reported as not detected.

[0058] In another preferred embodiment, the sample to be tested is selected from the group consisting of whole blood, plasma, serum, or a combination thereof.

[0059] In a fifth aspect of the present invention, a method for detecting bloodstream infection pathogens is provided, comprising the following steps:

[0060] (i) extracting free DNA from the sample to be tested;

[0061] (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;

[0062] (iii) Read the fluorescence data and calculate the copy concentration of the target sequence to obtain the detection result.

[0063] In another preferred embodiment, in step (iii), the determination of the test result is as follows:

[0064] (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;

[0065] (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;

[0066] (3) If there are 1 or 2 positive droplets, it is indicated as a gray area and re-testing is recommended; if the re-test still shows less than 3 positive droplets, it is determined to be a negative result and reported as not detected.

[0067] In another preferred embodiment, the sample to be tested is selected from the group consisting of whole blood, plasma, serum, or a combination thereof.

[0068] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0069] In another preferred embodiment, the method is an in vitro method.

[0070] 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

[0071] Figure 1 The figure shows the detection results of Pseudomonas aeruginosa positive detected using the multiple primer-probe combination of the present invention.

[0072] Figure 2 The figure shows the detection results of Acinetobacter baumannii positive detected using the multiple primer probe combination of the present invention.

[0073] Figure 3 The figure shows the detection results of Enterococcus positive detected by the multiple primer-probe combination of the present invention.

[0074] Figure 4 The figure shows the detection results of Streptococcus positive detected using the multiple primer-probe combination of the present invention.

[0075] Figure 5 The figure shows the detection result of the internal control positive detected by using the multiple primer-probe combination of the present invention.

[0076] Figure 6 Shown are the concentration results for amplification of P. aeruginosa target genes using multiplex primer-probe combinations.

[0077] Figure 7 Concentration results for singleplex amplification of the P. aeruginosa target gene are shown.

[0078] Figure 8 Shown are the concentration results for amplification of the target gene of Acinetobacter baumannii using a multiplex primer-probe combination.

[0079] Figure 9 Concentration results for singleplex amplification of the target gene of Acinetobacter baumannii are shown.

[0080] Figure 10 Shown are the concentration results for amplification of the Enterococcus (E. faecalis) target gene using a multiplex primer-probe combination.

[0081] Figure 11 Shown are concentration results for singleplex amplification of the Enterococcus (E. faecalis) target gene.

[0082] Figure 12 Shown are the concentration results for amplification of a Streptococcus (S. pneumoniae) target gene using a multiplex primer-probe combination.

[0083] Figure 13 Shown are concentration results for singleplex amplification of the Streptococcus (S. pneumoniae) target gene.

[0084] Figure 14 The detection sensitivity of Pseudomonas aeruginosa in Example 2 is shown.

[0085] Figure 15 The detection sensitivity of Acinetobacter baumannii in Example 2 is shown.

[0086] Figure 16 The detection sensitivity of Enterococcus (Enterococcus faecalis) in Example 2 is shown.

[0087] Figure 17 The detection sensitivity of Enterococcus (Enterococcus faecium) in Example 2 is shown.

[0088] Figure 18 The detection sensitivity of Enterococcus (Enterococcus leucoderma) in Example 2 is shown.

[0089] Figure 19 The detection sensitivity of Enterococcus spp. (Enterococcus durophilus) in Example 2 is shown.

[0090] Figure 20 The detection sensitivity of Enterococcus (small intestinal Enterococci) in Example 2 is shown.

[0091] Figure 21 The detection sensitivity of Streptococcus (Streptococcus pneumoniae) in Example 2 is shown.

[0092] Figure 22 The detection sensitivity of Streptococcus (Streptococcus mitis) in Example 2 is shown.

[0093] Figure 23 The detection sensitivity of Streptococcus (Streptococcus agalactiae) in Example 2 is shown.

[0094] Figure 24 The detection sensitivity of Streptococcus (Streptococcus sanguis) in Example 2 is shown.

[0095] Figure 25 The detection sensitivity of Streptococcus (Streptococcus salivarius) in Example 2 is shown.

[0096] Figure 26 The specific detection results of the mixed pathogens in Example 3 are shown. DETAILED DESCRIPTION

[0097] After extensive and in-depth research and extensive screening, the inventors unexpectedly developed primers and probes that can be used to rapidly detect bloodstream infection pathogens with high specificity and sensitivity. The bloodstream infection pathogens include: Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus, and Streptococcus. The Enterococci include: Enterococcus faecalis, Enterococcus faecium, Enterococcus leucovorus, Enterococcus duril, and Enterococcus spp.; the Streptococci include: Streptococcus pneumoniae, Streptococcus mitis, Streptococcus agalactiae, Streptococcus sanguinis, and Streptococcus salivarius. Experiments of the present invention have shown that the digital PCR method of the present invention has a minimum detection sensitivity of 5 copies / reaction and good specificity. On this basis, the present invention was completed.

[0098] the term

[0099] 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.

[0100] 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."

[0101] 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).

[0102] 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.

[0103] bloodstream infection

[0104] 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.

[0105] BSI has a 40% chance of developing into sepsis. China reports 6.12 million new cases of sepsis annually, with a 90-day mortality rate of 33.5%, an average ICU stay of 32 days, and an average treatment cost of 300,000 yuan. Studies have shown that for every hour of delay in treatment for patients with septic shock experiencing hypotension, survival decreases by 7.6%, reaching a median survival rate of 42% after a six-hour delay.

[0106] Currently, blood culture is the gold standard for laboratory diagnosis of BSI, but its positive rate is only about 10%, and it takes at least 2-3 days to report the culture 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 molecular detection methods are all based on positive blood culture cultures. There are also some mNGS-based detection methods in China, but the mNGS detection time is 1-2 days, and it is costly, complex to operate, and requires professional bioinformatics analysts. It is difficult to carry out widely in medical institutions, and none of them have obtained registration certificates from the China National Medical Products Administration (NMPA).

[0107] 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.

[0108] The multiplex digital PCR (dPCR) rapid detection method achieves absolute quantification of nucleic acid molecules by randomly dividing the fluorescent quantitative reaction system into tens of thousands of independent tiny reaction units and using Poisson distribution and the proportion of positive droplets. As a third-generation PCR technology, dPCR has higher sensitivity than fluorescent quantitative PCR and strong anti-interference ability, which can achieve absolute quantification. It is suitable for the highly challenging detection of BSI, which has low pathogen load levels, complex blood components, and high human background DNA signals. The 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 for blood culture to 4 hours. It will effectively solve the current global problems of low detection rates and long reporting cycles for bloodstream infection pathogens.

[0109] Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococci, and Streptococci

[0110] Pseudomonas aeruginosa is a Gram-negative bacillus found in a wide range of environments, including water, soil, and plants. It is highly adaptable and resistant to antibiotics, often leading to hospital-acquired infections, particularly in patients with weakened immune systems or those taking long-term antibiotics. Pseudomonas aeruginosa can cause severe bloodstream infections, commonly seen in burn victims, those in intensive care units (ICUs), and those with central venous catheters. Infections can lead to sepsis, carry a high mortality rate, and are difficult to treat due to the bacterium's inherent resistance to multiple antibiotics.

[0111] Acinetobacter baumannii is a Gram-negative bacillus widely distributed in both natural and hospital settings. Its strong adaptability and drug resistance make it a major pathogen causing nosocomial infections. Acinetobacter baumannii often enters the bloodstream through infected medical devices (such as ventilators and catheters) or wounds, leading to bloodstream infections. Acinetobacter baumannii infections are common in ICU patients, particularly those receiving long-term broad-spectrum antibiotics or immunosuppressants. Its multidrug resistance makes it challenging to treat.

[0112] Enterococci are Gram-positive cocci commonly found in the intestines of humans and animals. Common pathogenic species include Enterococcus faecalis and Enterococcus faecium. Enterococci can enter the bloodstream through intestinal translocation or medical devices (such as urinary catheters and central venous catheters), causing bloodstream infections. Enterococcal infections are common in patients with compromised immune systems, prolonged hospitalizations, or those receiving broad-spectrum antibiotic therapy.

[0113] Streptococci are Gram-positive cocci that are widely present in the human respiratory, digestive, and genitourinary tracts. Streptococci can spread from local infections (such as respiratory and skin infections) to the bloodstream, causing bloodstream infections. Streptococcus pneumoniae is a common pathogen of community-acquired bloodstream infections, while Streptococcus pyogenes can cause severe invasive infections (such as necrotizing fasciitis) and lead to sepsis.

[0114] Detection method of the present invention

[0115] The present invention provides a method for detecting pathogens that cause bloodstream infection, the method comprising the following steps:

[0116] 1) Based on the gene sequence of the pathogen, specific primer probes (including primer and probe sequences for internal reference) are designed. The pathogens include Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus and Streptococcus. The Enterococcus genus includes: Enterococcus faecalis, Enterococcus faecium, Enterococcus leucoderma, Enterococcus durophilus and Enterococcus spp.; the Streptococci include: Streptococcus pneumoniae, Streptococcus mitis, Streptococcus agalactiae, Streptococcus sanguinis and Streptococcus salivarius. The above primers and probes are used for digital PCR detection, and the optimal detection conditions of the PCR system are optimized. The specific primer and probe sequences are shown in Table 1 below.

[0117] Table 1

[0118]

[0119] 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.

[0120] 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 a blood sample, the steps are as follows:

[0121] 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.

[0122] Second, separation of plasma: The first step is to remove cells by low-speed centrifugation at 4°C, 1600g, and 10 minutes.

[0123] Third, extraction: Use a plasma free nucleic acid extraction kit and follow the instructions for extraction.

[0124] 3) Digital PCR.

[0125] First, configure the PCR system. In the reagent preparation area, configure the PCR system according to the following table:

[0126] 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.

[0127] Preferably, the final concentration of the primers is 0.33 μM each, and the final concentration of the probes is 0.17 μM each.

[0128] Second, add the template. Add the 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 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.

[0129] Third, generate droplets. Generate droplets according to the instrument requirements.

[0130] 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 (95°C for 30 seconds, 56°C for 15 seconds, and 72°C for 15 seconds), with a ramp rate of 2°C / s.

[0131] Fifth, scan. Based on the scan results, determine whether the sample is positive or negative.

[0132] The test results are determined as follows:

[0133] (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;

[0134] (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;

[0135] (3) If there are 1 or 2 positive droplets, it is indicated as a gray area and re-testing is recommended; if the re-test still shows less than 3 positive droplets, it is determined to be a negative result and reported as not detected.

[0136] The main advantages of the present invention include:

[0137] (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 solve problems such as low sensitivity, poor specificity, high requirements for sample type and quality, and complex positive interpretation methods.

[0138] (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, offering a sensitivity advantage unmatched by other technologies. The detection method of the present invention has been validated to achieve a minimum detection limit of 5 copies / reaction.

[0139] (c) Strong specificity: The designed specific primers and probes are targeted at the specific sequences of Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus, and Streptococcus, respectively, and can specifically amplify the target location.

[0140] (d) The primer-probe combination of the present invention has relaxed requirements for sample type and quality and is highly resistant to interference. Due to its high sensitivity, the present invention is suitable for peripheral blood samples (which are relatively easy to obtain but have low DNA content and are fragmented). Furthermore, due to the uniqueness of its digital PCR platform, it can divide the reaction system into approximately 20,000 small systems and also divide interfering substances into approximately 20,000 parts, greatly reducing the impact of interfering substances on the reaction and, of course, allowing the detection of samples with more complex backgrounds. This is something that other platforms cannot achieve.

[0141] (e) Simple positive interpretation: Since the present invention uses an absolute quantitative method, there is no need to set up a control standard curve. The results can be used to determine whether the target mutant template is present based on the two-dimensional fluorescence graph. The interpretation method is as follows:

[0142] (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;

[0143] (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;

[0144] (3) If there are 1 or 2 positive droplets, it is indicated as a gray area and re-testing is recommended; if the re-test still shows less than 3 positive droplets, it is determined to be a negative result and reported as not detected.

[0145] 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 based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0146] Example 1 Digital PCR Detection of Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus, and Streptococcus

[0147] 1.1 Primer and probe design

[0148] Design of primers and probes for pathogen detection: Multiple sets of primers and probes were designed based on the specific genes of Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus, and Streptococcus (Table A). Finally, sequences with no mutual influence and good amplification efficiency were screened out, as shown in Table 2 below.

[0149] 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.

[0150] Table 2

[0151]

[0152] Table A Specific genes and their sequence information of Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus and Streptococcus

[0153]

[0154]

[0155]

[0156] 1.2 PCR system

[0157] 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.

[0158] 1.3 PCR program: 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s).

[0159] 1.4 Chip Scanning

[0160] After testing, it can effectively detect Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus (Enterococcus faecalis), Streptococcus (Streptococcus pneumoniae) and internal reference, respectively. Figure 1-Figure 5 shown.

[0161] In addition, five common clinical samples of Enterococcus were tested, and the test results are shown in Table 3 below.

[0162] Table 3

[0163] Serial number Identification of pathogens Detection 1 Enterococcus faecalis Checkout 2 Enterococcus faecium Checkout 3 Enterococcus leucoderma Checkout 4 enterococci Checkout 5 Enterococci Checkout

[0164] Four common clinical samples of Streptococcus were tested, and the test results are shown in Table 4 below.

[0165] Table 4

[0166] Serial number Identification of pathogens Detection 1 Streptococcus pneumoniae Checkout 2 Streptococcus mitis Checkout 3 Streptococcus agalactiae Checkout 4 Streptococcus sanguis Checkout 5 Streptococcus salivarius Checkout

[0167] By comparing the single and multiplex results for each target, it can be seen that there is no difference in the amplification concentration results between single and multiplex. Therefore, there is no mutual influence between the primers and probes that leads to a decrease in PCR amplification efficiency. Figure 6-Figure 13 and Table 5.

[0168] Table 5

[0169]

[0170] Example 2: Sensitivity Verification of Digital PCR for Detection of Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus, and Streptococcus

[0171] 2.1 Experimental methods

[0172] The copy number (copies / μL) of each pathogen template was calculated according to Example 1, and each pathogen template was diluted to 8 copies / μL, 4 copies / μL, 2 copies / μL, and 1 copy / μL using TE to prepare a gradient dilution template.

[0173] Verification 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.

[0174] Add template to the sample preparation area in the following order: blank control, negative control, and serially diluted template. The blank control is water, the negative control is tgDNA, and the serially diluted template is 8 copies / μL, 4 copies / μL, 2 copies / μL, and 1 copy / μL of each pathogen genome.

[0175] The PCR reaction system was generated into droplets in the same manner as in Example 1. PCR was performed according to the PCR program: 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s). Plate reading was started according to the instrument requirements.

[0176] 2.2 Experimental Results

[0177] Digital PCR results are attached. Figure 14-Figure 25The 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 can effectively detect positive spots at 1 copy / μL of template, meaning the minimum detection sensitivity of the digital PCR method of the present invention is 5 copies / reaction.

[0178] Example 3 Specificity Verification of Digital PCR Detection of Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus, and Streptococcus

[0179] 3.1 Experimental methods

[0180] Primers and probes for different pathogens were designed. The copy number (copies / μL) of each pathogen template was calculated according to Example 1. Each pathogen template was diluted to 10,000 copies / μL with TE. The pathogens were then mixed in equal proportions in groups of five (a total of seven groups or seven samples), as shown in Table 6 below.

[0181] Table 6

[0182]

[0183] Verification 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.

[0184] 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 6 above.

[0185] The PCR reaction system was generated into droplets in the same manner as in Example 1. PCR was performed according to the PCR program: 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s). Plate reading was started according to the instrument requirements.

[0186] 3.2 Experimental Results

[0187] Digital PCR results are shown in Figure 26 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.

[0188] 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 these equivalents also fall within the scope defined in the appended documents.

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 Pseudomonas aeruginosa as shown in SEQ ID NOs: 1 to 3; (b) upstream and downstream primers and probes for detecting Acinetobacter baumannii as shown in SEQ ID NOs: 4 to 6; (c) upstream and downstream primers and probes for detecting Enterococcus as shown in SEQ ID NOs: 7 to 9; and (d) Upstream and downstream primers and probes for detecting Streptococcus as shown in SEQ ID NOs: 10 to 12.

2. The primer-probe combination according to claim 1, wherein The Enterococcus genus includes: Enterococcus faecalis, Enterococcus faecium, Enterococcus leucoflavus, Enterococcus durophilus and Enterococcus spp.; the Streptococcus genus includes: Streptococcus pneumoniae, Streptococcus mitis, Streptococcus agalactiae, Streptococcus sanguinis and Streptococcus salivarius.

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: 13-15.

4. The primer-probe combination according to claim 1, wherein The fluorescent reporter group of the Pseudomonas aeruginosa probe is FAM; the fluorescent reporter group of the Acinetobacter baumannii probe is HEX; the fluorescent reporter group of the Enterococcus probe is ROX; and the fluorescent reporter group of the Streptococcus 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: Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus and Streptococcus.

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 Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus and Streptococcus genomes; the negative quality control product is normal human leukocyte genomic DNA (tgDNA).

9. Use of the primer-probe combination according to claim 1 or the kit according to claim 6, characterized in that: Used to prepare reagents for detecting bloodstream infection pathogens.

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 or the kit of claim 6; (iii) Read the fluorescence data and calculate the copy concentration of the target sequence to obtain the detection result.

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