Method and kit for detecting bloodstream infection pathogens and application

Abstract

The invention provides a method and a kit for detecting blood flow infection pathogens and application of the method and the kit. Specifically, the invention provides a primer probe combination for detecting bloodstream infection pathogens, and the primer probe combination comprises: (a) upstream and downstream primers and probes for detecting serratia marcescens as shown in SEQ ID NO: 7-9; (b) an upstream primer, a downstream primer and a probe which are shown as SEQ ID NO: 10-12 and are used for detecting burkholderia cepacia; (c) an upstream primer, a downstream primer and a probe for detecting stenotrophomonas maltophilia as shown in SEQ ID NO: 1-3; and (d) upstream and downstream primers and a probe which are shown as SEQ ID NO: 4-6 and are used for detecting enterobacter cloacae. 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 diagnostics, and specifically, to a method, a kit, and an application for detecting pathogens causing bloodstream infection. Background Art

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

[0003] Due to factors such as the increase in invasive procedures, the increasing use of broad-spectrum antibacterial drugs and corticosteroid drugs, the incidence and mortality of bloodstream infection have been rising year by year. After the occurrence of bloodstream infection, the prognosis may be poor, resulting in an extended hospital stay, thereby increasing the economic burden on patients. The earlier the pathogen causing bloodstream infection is identified and the correct antibacterial drug treatment is selected, the mortality of patients will decrease by about 7%. Therefore, it is crucial to quickly and timely identify the pathogenic bacteria causing bloodstream infection.

[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. Currently, the main detection methods for BSI are: (1) blood culture, but it has deficiencies such as low detection rate, long detection time, high contamination rate, 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 cautious when used to identify Gram-positive bacteria (about 80% consistent with subsequent culture results); (3) next-generation sequencing technology (NGS), the detection cost is high, so it is not suitable for the rapid detection required by general bloodstream infection patients, and NGS needs to first enrich the DNA of the detected sample by PCR, so there will be amplification preference for some regions, resulting in missed detection and insufficient accuracy.

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

[0006] The present invention provides a method and a kit for detecting bloodstream infection that are rapid, highly specific, highly sensitive, and have strong anti-interference ability.

[0007] In the first aspect of the present invention, a primer-probe combination (or primer-probe composition) for detecting pathogens causing bloodstream infection is provided, and the primer-probe combination (or primer-probe composition) includes: (a) Forward and reverse primers and a probe for detecting Serratia marcescens as shown in SEQ ID NOs: 7 - 9.

[0008] In another preferred embodiment, the primer - probe combination further comprises a primer - probe combination selected from the following group: (b) Forward and reverse primers and a probe for detecting Burkholderia cepacia as shown in SEQ ID NOs: 10 - 12; (c) Forward and reverse primers and a probe for detecting Stenotrophomonas maltophilia as shown in SEQ ID NOs: 1 - 3; (d) Forward and reverse primers and a probe for detecting Enterobacter cloacae as shown in SEQ ID NOs: 4 - 6.

[0009] (e) At least two primer - probe combinations among (b) - (d) above.

[0010] In a preferred aspect of the present invention, a primer - probe combination for detecting blood - stream infection pathogens is provided, and the primer - probe combination comprises: (a) Forward and reverse primers and a probe for detecting Serratia marcescens as shown in SEQ ID NOs: 7 - 9; (b) Forward and reverse primers and a probe for detecting Burkholderia cepacia as shown in SEQ ID NOs: 10 - 12; (c) Forward and reverse primers and a probe for detecting Stenotrophomonas maltophilia as shown in SEQ ID NOs: 1 - 3; and (d) Forward and reverse primers and a probe for detecting Enterobacter cloacae as shown in SEQ ID NOs: 4 - 6.

[0011] In another preferred embodiment, the primer - probe combination further comprises primers and a probe for detecting other blood - stream infection pathogens.

[0012] In another preferred embodiment, the primer - probe combination and the primer - probe composition have the same meaning and can be used interchangeably.

[0013] In another preferred embodiment, the primer - probe combination further comprises: (e) Forward and reverse primers and a probe for detecting an internal reference (or internal reference gene) as shown in SEQ ID NOs: 17 - 19.

[0014] In another preferred embodiment, the primers and probe for detecting the internal reference are designed for a specific conserved region of the human EGFR gene.

[0015] In this text, "mutually distinct and non-interfering" means that the fluorescent groups used for each probe in the composition are different and do not affect the detection of each other, that is, they can be detected using different channels. For example, FAM, HEX, ROX, CY5, and Q705 can be used. The absorption values of these groups are not close, and different channels can be selected, so they do not interfere with each other.

[0016] In another preferred example, 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; the fluorescent reporter group of the Enterobacter cloacae probe is HEX.

[0017] In another preferred example, the fluorescent reporter group of the internal reference probe is Q705.

[0018] In another preferred example, the 3'-end of the probe also has a fluorescent quenching group.

[0019] In another preferred example, the fluorescent quenching group is selected from the following group: BHQ1, BHQ2, BHQ3.

[0020] In another preferred example, the fluorescent quenching groups at the 3'-ends of the Stenotrophomonas maltophilia probe and the Enterobacter cloacae probe are BHQ1; the fluorescent quenching groups at the 3'-ends of the Serratia marcescens probe and the Burkholderia cepacia probe are BHQ2.

[0021] In another preferred example, the fluorescent quenching group at the end of the internal reference probe is BHQ3.

[0022] In another preferred example, the components of the primer-probe combination are each present in a separate package.

[0023] In another preferred example, the components of the primer-probe combination are present in the same package.

[0024] In another preferred example, the components of the combination are present in a mixed form.

[0025] In another preferred example, the 5'-end of the probe sequence is linked to a fluorescent reporter gene, and the 3'-end is linked to a fluorescent quenching group.

[0026] In the second aspect of the present invention, there is provided the use of the primer-probe combination as described in the first aspect of the present invention for preparing a kit for detecting blood-stream infection pathogens; wherein, the pathogens include: Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

[0027] In a third aspect of the present invention, a kit for detecting blood stream infection pathogens is provided, and the kit comprises the primer-probe combination as described in the first aspect of the present invention.

[0028] In another preferred embodiment, the kit further comprises a negative control and a positive control.

[0029] In another preferred embodiment, the negative control is at least one of DEPC-H2O, physiological saline, internal reference gene pseudovirus, and normal human leukocyte genomic DNA.

[0030] In another preferred embodiment, the negative control is normal human leukocyte genomic DNA (tgDNA).

[0031] In another preferred embodiment, the copy number concentration of the tgDNA is 1000 copies / μL.

[0032] In another preferred embodiment, the kit further comprises: (e) upstream and downstream primers and a probe for detecting an internal reference as shown in SEQ ID NOs: 17-19.

[0033] In another preferred embodiment, the positive control is at least one of fragment plasmids or fragment DNAs of Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

[0034] In another preferred embodiment, the positive control is a mixture of the genomes of Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

[0035] In another preferred embodiment, the copy number of all target sites in the positive control is 50 copies / μL.

[0036] In another preferred embodiment, the kit further comprises a PCR reaction solution.

[0037] In another preferred embodiment, the PCR reaction solution comprises at least one of dNTP, PCR buffer, Mg 2+ and DNA polymerase.

[0038] In another preferred embodiment, the DNA polymerase is 5×HS Taq Buffer with Mg 2+ reaction system.

[0039] In another preferred embodiment, the kit further comprises an instruction manual, and the instruction manual indicates that the kit is used for detecting blood stream infection pathogens.

[0040] In another preferred embodiment, the kit further comprises a blank control, and the blank control is ultrapure water.

[0041] In a fourth aspect of the present invention, there is provided the use of the primer-probe combination as described in the first aspect of the present invention or the kit as described in the third aspect of the present invention for preparing a reagent for detecting blood-stream infection pathogens.

[0042] In another preferred embodiment, the detection comprises the following steps: (i) Extracting free DNA from the sample to be tested; (ii) Performing fluorescence quantitative PCR on the free DNA obtained in step (i) using the primer-probe combination as described in the first aspect of the present invention or the kit as described in the third aspect of the present invention; (iii) Reading the fluorescence data and calculating the copy concentration of the target sequence, thereby obtaining the detection result.

[0043] In another preferred embodiment, in step (iii), the determination of the detection result is as follows: (1) If the number of positive droplets ≥ 3 and the clustering is consistent with the positive control, it indicates a positive result, and the infecting pathogen and the copy number are directly reported; (2) If there are no positive droplets and the clustering is consistent with the negative control, it indicates a negative result, and it is reported that no detection is found; (3) If there is 1 or 2 positive droplets, it indicates a gray zone, and re-examination is recommended; if there are still < 3 positive micro-droplets after re-examination, it is determined as a negative result, and it is reported that no detection is found.

[0044] In another preferred embodiment, the sample to be tested is selected from the group consisting of: whole blood, plasma, serum, or a combination thereof.

[0045] In another preferred embodiment, the blood-stream infection pathogens include: Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

[0046] In a fifth aspect of the present invention, there is provided a method for detecting blood-stream infection pathogens, comprising the following steps: (i) Extracting free DNA from the sample to be tested; (ii) Performing fluorescence quantitative PCR on the free DNA obtained in step (i) using the primer-probe combination as described in the first aspect of the present invention or the kit as described in the third aspect of the present invention; (iii) Reading the fluorescence data and calculating the copy concentration of the target sequence, thereby obtaining the detection result.

[0047] In another preferred embodiment, the blood-stream infection pathogens include: Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae.

[0048] In another preferred embodiment, in step (iii), the determination of the detection result is 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 infected pathogen and the copy number are directly reported; (2) If there are no positive droplets and the clustering is consistent with the negative control, it indicates a negative result, and it is reported that no detection is found; (3) If there is 1 or 2 positive droplets, it indicates a gray zone, and retesting is recommended; if there are still <3 positive micro-droplets after retesting, it is determined as a negative result, and it is reported that no detection is found.

[0049] In another preferred example, the sample to be tested is selected from the following group: whole blood, plasma, serum, or a combination thereof.

[0050] In another preferred example, the method is non-diagnostic and non-therapeutic.

[0051] In another preferred example, the method is an in vitro method.

[0052] 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 specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The figure shows the detection result diagram of Stenotrophomonas maltophilia detected by multiplex digital PCR using the primer-probe combination of the present invention.

[0054] Figure 2 The figure shows the detection result diagram of Enterobacter cloacae detected by multiplex digital PCR using the primer-probe combination of the present invention.

[0055] Figure 3 The figure shows the detection result diagram of Serratia marcescens detected by multiplex digital PCR using the primer-probe combination of the present invention.

[0056] Figure 4 The figure shows the detection result diagram of Burkholderia cepacia detected by multiplex digital PCR using the primer-probe combination of the present invention.

[0057] Figure 5 The figure shows the detection result diagram of the internal reference detected by multiplex digital PCR using the primer-probe combination of the present invention.

[0058] Figure 6 The figure shows the detection sensitivity of Stenotrophomonas maltophilia in Example 2.

[0059] Figure 7 The figure shows the detection sensitivity of Enterobacter cloacae in Example 2.

[0060] Figure 8 Shows the detection sensitivity of Serratia marcescens in Example 2.

[0061] Figure 9 Shows the detection sensitivity of Burkholderia cepacia in Example 2.

[0062] General description of the drawings: The gray markings present in the drawings represent supplementary explanations of the figures, used to prompt the specific single-channel fluorescence information of the individual figures below, that is, the signal intensity measured by the droplets in a certain specific fluorescence detection channel (such as FAM, HEX, ROX, CY5). Detailed implementation manners

[0063] Through extensive and in-depth research and a large number of screenings, the present 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: Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae. Experiments of the present invention show that the lowest detection sensitivity of the digital PCR method of the present invention is 5 copies / reaction, and it has good specificity. On this basis, the present invention was completed.

[0064] Terms To make the present disclosure easier to understand, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0065] As used herein, the term "comprising" or "including" can be open-ended, semi-closed, and closed-ended. In other words, the term also includes "consisting essentially of...", or "consisting of...".

[0066] As used herein, unless otherwise specified, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional values (such as one-tenth and one-hundredth of an integer).

[0067] As used herein, the term "and / or" relates to and encompasses any and all possible combinations of one or more of the related listed items.

[0068] Bloodstream infection Bloodstream infection (BSI) refers to an infection caused by pathogenic microorganisms entering the blood circulation, and the main pathogens include bacteria, fungi, and viruses, etc. BSI has the typical characteristics of three highs and one difficulty: high incidence, high mortality, high treatment cost, and difficult diagnosis.

[0069] BSI has a 40% probability of developing into sepsis. There are 6.12 million new cases of sepsis in China every year, with a 90-day mortality rate of 33.5%, an average length of stay in the ICU of 32 days, and an average treatment cost of 300,000 yuan. The study found that for septic shock patients, the survival rate decreased by 7.6% for every 1-hour delay in treatment after hypotension, and the survival rate for a median delay of 6 hours was 42%.

[0070] 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 FilmArray BCID from bioMérieux, Verigene Gram+ BC and Verigene Gram- BC from Luminex. However, these molecular detection methods are all based on positive blood culture isolates for detection. There are also some mNGS-based detection methods in China, but the mNGS detection time is 1 - 2 days, and it has high costs, complex operations, and requires professional bioinformatics analysts, making it difficult to be widely carried out in medical institutions, and none of them have obtained registration certificates from the National Medical Products Administration.

[0071] The long time-consuming and difficult diagnosis of BSI pathogen in the laboratory is a major challenge that urgently needs to be solved in current clinical emergency departments and infection departments, and it highly endangers people's lives and health. Therefore, a blood stream infection pathogen detection method with high sensitivity and fast detection speed will help quickly identify the cause, reduce mortality, effectively improve the timeliness of diagnosis and treatment, enhance the medical level, and improve medical efficiency.

[0072] The multiplex digital PCR (dPCR) rapid detection method randomly divides the fluorescence quantitative reaction system into tens of thousands of independent tiny reaction units, and realizes the absolute quantification of nucleic acid molecules according to the Poisson distribution and the proportion of positive droplets. As the third-generation PCR technology, dPCR has higher sensitivity than fluorescence quantitative PCR, strong anti-interference ability, can achieve absolute quantification, and is suitable for such difficult detections as BSI with low levels of pathogenic microorganisms, complex blood components, and high human background DNA signals. The innovative rapid blood stream 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 of blood culture to 4 hours, which will effectively solve the worldwide problems such as low detection rate and long reporting cycle of current blood stream infection pathogens.

[0073] Serratia marcescens, Burkholderia cepacia complex, Stenotrophomonas maltophilia and Enterobacter cloacae Serratia marcescens is a Gram-negative bacillus and belongs to the Enterobacteriaceae family. It can enter the bloodstream through medical devices (such as catheters) or surgical wounds, causing bloodstream infections (bacteremia). It is commonly found in immunocompromised patients, especially those with long-term hospital stays or undergoing invasive treatments. The infection may lead to sepsis and be life-threatening in severe cases.

[0074] Burkholderia cepacia is a Gram-negative bacillus and is widely present in soil and water. It can enter the bloodstream through respiratory infections or medical devices (such as catheters), causing bloodstream infections. It has natural resistance to a variety of antibiotics, making treatment difficult, and may lead to sepsis or multiple organ failure.

[0075] Stenotrophomonas maltophilia is a Gram-negative bacillus and is widely present in water, soil, and hospital environments. It can enter the bloodstream through medical devices (such as catheters) or respiratory infections, causing bloodstream infections. It has natural resistance to a variety of antibiotics, making treatment difficult, and may lead to sepsis or septic shock.

[0076] Enterobacter cloacae is a Gram-negative bacillus and belongs 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 a variety of antibiotics, especially strains producing extended-spectrum β-lactamases (ESBLs), and may lead to sepsis or septic shock.

[0077] In summary, the above bacteria are all opportunistic pathogens, commonly found in hospital environments, especially in immunocompromised patients or those undergoing invasive treatments. The treatment of bloodstream infections requires selecting appropriate antibiotics based on the results of pathogen detection.

[0078] The detection method of the present invention The present invention provides a method for detecting pathogens causing bloodstream infections, and the bloodstream infection pathogens include Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae. The specific experimental steps are as follows: 1) According to the gene sequences of the pathogens, specific primer-probes (including internal reference primers and probes) are designed. The pathogens include Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae. Digital PCR detection is carried out using the above primers and probes, and the optimal detection conditions of the PCR system are optimized. This method is applied to detect patient blood samples. The specific primer-probe sequences are shown in Table 1.

[0079] Table 1

[0080] Among them, the 5' end of the probe sequence is also connected to a fluorescent reporter gene, and the 3' end is also connected to a fluorescent quenching group. The fluorescent reporter group is selected from the following group: FAM, HEX, ROX, CY5, and Q705; the fluorescent quenching group is selected from the following group: BHQ1, BHQ2, BHQ3.

[0081] Perform digital PCR detection using the above primers and probes, and optimize the best detection conditions for the PCR system.

[0082] 2) Extract the nucleic acid of the sample. Select a method suitable for the sample type to extract nucleic acid, and the sample type includes peripheral blood, peripheral blood plasma / serum. In particular, for the extraction of cell-free nucleic acid from blood samples, the steps are as follows: First, the selection of blood collection tubes. If a common blood collection tube is used, it cannot contain heparin, and plasma needs to be separated within 4 hours. If the blood cannot be processed in time, a blood collection tube with blood cell stabilization technology needs to be selected.

[0083] Second, the separation of plasma. Separation of plasma: The first step is to centrifuge at low speed to remove cells, at 4°C, 1600g, for 10 minutes.

[0084] Third, extraction. Use a special extraction kit for plasma cell-free nucleic acid and extract according to the instructions.

[0085] 3) Digital PCR.

[0086] First, configure the PCR system. Configure the PCR system in the reagent preparation area according to the following table: 5×HS Taq Buffer with Mg 2+ 6 μL, dNTPs (10 mM each) 0.75 μL, HotStart TaqDNA Polymerase 0.2 μL, SEQ ID NO: 1 (10 μM) 1 μL, SEQ ID NO: 2 (10 μM) 1 μL, SEQ ID NO:3 (10 μM) 0.5 μL, SEQ ID NO: 4 (10 μM) 1 μL, 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.

[0087] Preferably, the final concentration of each primer is 0.33 μM, and the final concentration of each probe is 0.17 μM.

[0088] Second, add the template. Add the template in the following order in the sample preparation area: the sample to be tested, blank control, negative control, and positive control. The negative control is normal human leukocyte genomic DNA (abbreviated as "tgDNA") with a copy concentration of 1000 copies / μL, and the copy number of all target sites in the positive control solution is 50 copies / μL. The blank control is ultrapure water.

[0089] Third, generation of droplets. Generate droplets according to the instrument requirements.

[0090] Fourth, PCR. After the annealing temperature optimization experiment, it was found that the digital PCR effect was optimal with annealing at 56°C for 15 s. Perform PCR according to the following program: 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s), and the heating and cooling rate is 2°C / s.

[0091] Fifth, read the plate. Judge whether the sample is positive or negative according to the read plate result.

[0092] The determination of the detection result is as follows: (1) If the number of positive droplets ≥ 3 and the clustering is consistent with the positive control, it indicates a positive result, and directly report the infected pathogen and the copy number; (2) If there are no positive droplets and the clustering is consistent with the negative control, it indicates a negative result, and report that it is not detected; (3) If there is 1 or 2 positive droplets, it indicates a gray area, and it is recommended to retest; if there are still < 3 positive droplets after retesting, it is determined as a negative result, and report that it is not detected.

[0093] The kit of the present invention The present invention provides a kit for detecting Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae. The kit includes PCR primer probes, reaction systems, reaction conditions, negative control DNA samples, and positive control DNA samples. The following content: Primers and probes (including internal reference primers and probes): Synthesize the oligonucleotide fragments and modified oligonucleotide fragments shown in Table 1, and dissolve them in TE (10 μM), as specifically described in the foregoing part.

[0094] The reaction conditions are specifically described in the foregoing part.

[0095] PCR reagent: The DNA polymerase uses 5×HS Taq Buffer with Mg 2+ The reaction system is as described in the foregoing part.

[0096] Control: The blank control is water, the negative control is the genomic DNA of normal human white blood cells (tgDNA) with a copy concentration of 1000 copies / μL. The positive control is a mixture of the genomic DNA of Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococcus, and Staphylococcus aureus. The copy number of all target sites in the positive control solution is 50 copies / μL.

[0097] The main advantages of the present invention include: (a) Compared with the existing technologies such as blood culture, MALD-TOF MS, and NGS, the present invention uses Taqman probes in combination with digital PCR, which can solve the problems of low sensitivity, poor specificity, high requirements for the type and quality of samples, and complex positive interpretation methods.

[0098] (b) High sensitivity: Since this method uses a digital PCR platform, the reaction system can be divided into approximately 20,000 tiny reactions, and theoretically, single-copy mutations can be detected, with a sensitivity advantage that cannot be matched by other technologies. The detection method of the present invention can achieve a minimum detection limit of 5 copies / reaction through verification.

[0099] (c) Strong specificity: The designed specific primer probes are respectively directed against the specific sequences of Stenotrophomonas maltophilia, Enterobacter cloacae, Serratia marcescens, and Burkholderia cepacia, and can specifically amplify the target positions.

[0100] (d) The primer-probe combination of the present invention has loose requirements for the type and quality of samples and strong anti-interference ability. Due to the high sensitivity of the present invention, the applicable sample type is peripheral blood samples (such samples are relatively easy to obtain, but have low DNA content and are fragmented); moreover, due to the uniqueness of its digital PCR platform, that is, the reaction system can be divided into approximately 20,000 small systems, and at the same time, the interfering substances can also be divided into approximately 20,000 parts, which can greatly reduce the influence of interfering substances on the reaction, and of course, samples with a more complex background can be detected. This cannot be achieved by other platforms.

[0101] (e) The positive interpretation method of the present invention is simple: Since the present invention uses an absolute quantification method, there is no need to set a control standard curve, and whether the target mutant template is contained can be determined according to the two-dimensional fluorescence map. The interpretation method is as follows: (1) If the number of positive droplets ≥ 3, and the clustering is consistent with the positive control, it indicates a positive result, and the infectious pathogen and the copy number are directly reported; (2) If there are no positive droplets, and the clustering is consistent with the negative control, it indicates a negative result, and it is reported that no detection is found; (3) If there is 1 or 2 positive droplets, it indicates a gray area, and reexamination is recommended; if there are still < 3 positive droplets after reexamination, it is determined as a negative result, and it is reported that no detection is found.

[0102] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out 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 according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight percentage and weight parts.

[0103] Example 1 Detection of Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae by Digital PCR 1.1 Primer and Probe Design 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). Finally, the sequences with no mutual influence and excellent amplification efficiency were screened out as shown in Table 2 below. Among them, a fluorescent reporter gene was linked to the 5' end of the probe sequence during detection, and a fluorescent quenching group was linked to the 3' end.

[0104] Primer and probe design for internal reference: Primers and probes were screened from multiple human genes, aiming to have no mutual influence with the primers and probes for pathogen detection above and excellent amplification efficiency. Finally, the internal reference sequences in Table 2 below were obtained as the internal reference sequences of the kit. The upstream and downstream primers and probes for internal reference detection were designed for specific conserved regions of the human EGFR gene.

[0105] Table 2

[0106] Table A Specific Genes and Their Sequence Information of Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae

[0107]

[0108]

[0109] 1.2 PCR System 5×HS Taq Buffer with Mg 2+6 μL, 0.75 μL of dNTPs (10 mM each), 0.2 μL of HotStart Taq DNA Polymerase, 1 μL of SEQ ID NO: 1 (10 μM), 1 μL of SEQ ID NO: 2 (10 μM), 0.5 μL of SEQ ID NO: 3 (10 μM), 1 μL of SEQ ID NO: 4 (10 μM), 1 μL of SEQ ID NO: 5 (10 μM), 0.5 μL of SEQ ID NO: 6 (10 μM), 1 μL of SEQ ID NO: 7 (10 μM), 1 μL of SEQ ID NO: 8 (10 μM), 0.5 μL of SEQ ID NO: 9 (10 μM), 1 μL of SEQ ID NO: 10 (10 μM), 1 μL of SEQ ID NO: 11 (10 μM), 0.5 μL of SEQ ID NO: 12 (10 μM), 5 μL of template, and make up to 30 μL with water.

[0110] 1.3 PCR procedure: 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s).

[0111] 1.4 Chip scanning After testing, Stenotrophomonas maltophilia, Enterobacter cloacae, Serratia marcescens, and Burkholderia cepacia can be effectively detected, as Figures 1 - 5 shown.

[0112] Example 2 Verification of the sensitivity of digital PCR for the detection of Stenotrophomonas maltophilia, Enterobacter cloacae, Serratia marcescens, and Burkholderia cepacia 2.1 Experimental method Calculate the template copy number (copies / μL) of each pathogen according to Example 1, and dilute each pathogen template to 2 copies / μL and 1 copy / μL with TE to make gradient-diluted templates.

[0113] Verify the digital PCR system: 5×HS Taq Buffer with Mg 2+6 μL, 0.75 μL of dNTPs (10 mM each), 0.2 μL of HotStart Taq DNA Polymerase, 1 μL of SEQ ID NO: 1 (10 μM), 1 μL of SEQ ID NO: 2 (10 μM), 0.5 μL of SEQ ID NO: 3 (10 μM), 1 μL of SEQ ID NO: 4 (10 μM), 1 μL of SEQ ID NO: 5 (10 μM), 0.5 μL of SEQ ID NO: 6 (10 μM), 1 μL of SEQ ID NO: 7 (10 μM), 1 μL of SEQ ID NO: 8 (10 μM), 0.5 μL of SEQ ID NO: 9 (10 μM), 1 μL of SEQ ID NO: 10 (10 μM), 1 μL of SEQ ID NO: 11 (10 μM), 0.5 μL of SEQ ID NO: 12 (10 μM), 5 μL of template, and make up to 30 μL with water.

[0114] In the sample preparation area, add the gradient-diluted templates in the following order: 2 copies / μL and 1 copy / μL of each pathogen genome.

[0115] Generate droplets for the PCR reaction system in the same manner as in Example 1. Perform PCR 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). Start reading the plate according to the instrument requirements.

[0116] 2.2 Experimental Results The results of digital PCR are shown in Figures 6 - 9 In addition, the digital PCR method of the present invention can effectively detect positive points when detecting a 1 copy / μL template, that is, the lowest detection sensitivity of the digital PCR method of the present invention is 5 copies / reaction.

[0117] Specific verification of the digital PCR method for detecting Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia, and Enterobacter cloacae in Example 3 3.1 Experimental Method According to the types of clinical pathogen infections, purchase the following strains, extract the genomic DNA of the strains, and after extraction, perform digital PCR detection in the manner of Example 1 with a loading amount of 0.1 ng. The results are shown in Table 3 below.

[0118] Table 3

[0119]

[0120] 3.2 Experimental Results The results showed that only Stenotrophomonas maltophilia, Enterobacter cloacae, Serratia marcescens, and Burkholderia cepacia could be effectively detected, while the other strains could not be detected. That is, the digital PCR method of the present invention has good specificity.

[0121] All documents mentioned in the present invention are incorporated herein by reference as if each individual document was specifically and individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A primer-probe combination for detecting blood-stream infection pathogens, characterized in that, The primer-probe combination includes: (a) Upstream and downstream primers and a probe for detecting Serratia marcescens as shown in SEQ ID NOs: 7-9; (b) Upstream and downstream primers and a probe for detecting Burkholderia cepacia as shown in SEQ ID NOs: 10-12; (c) Upstream and downstream primers and a probe for detecting Stenotrophomonas maltophilia as shown in SEQ ID NOs: 1-3; and (d) Upstream and downstream primers and a probe for detecting Enterobacter cloacae as shown in SEQ ID NOs: 4-6.

2. The primer-probe combination according to claim 1, wherein The primer-probe combination further includes: (e) Upstream and downstream primers and a probe for detecting a reference gene as shown in SEQ ID NOs: 17-19.

3. The primer-probe combination according to claim 1, wherein 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 according to claim 1, wherein The fluorescent quenching group of both the Serratia marcescens probe and the Burkholderia cepacia probe is BHQ2; the fluorescent quenching group of the Stenotrophomonas maltophilia probe and the Enterobacter cloacae probe is BHQ1.

5. Use of the primer-probe combination according to claim 1, characterized in that, For preparing a kit for detecting blood stream infection pathogens; wherein, the pathogens include: Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia and Enterobacter cloacae.

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

7. The kit according to claim 6, characterized in that, The kit further includes a negative control and a positive control.

8. The kit according to claim 7, wherein The positive control is a mixture of the genomes of Serratia marcescens, Burkholderia cepacia, Stenotrophomonas maltophilia and Enterobacter cloacae; the negative control is genomic DNA of normal human leukocytes (tgDNA).

9. Use of the primer-probe combination according to claim 1 or the kit according to claim 6, characterized in that, For preparing a reagent for detecting blood stream infection pathogens.

10. The use according to claim 9, characterized in that, The detection includes the following steps: (i) Extracting free DNA from the sample to be tested; (ii) Performing fluorescence quantitative PCR on the free DNA obtained in step (i) using the primer-probe combination as described in claim 1 or the kit as described in claim 3; (iii) Reading the fluorescence data and calculating the copy concentration of the target sequence, thereby obtaining the detection result.

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