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 bloodstream infection pathogens, and the primer probe combination comprises: (a) upstream and downstream primers and probes for detecting coagulase negative staphylococcus as shown in SEQ ID NO: 7-9; (b) upstream and downstream primers and probes for detecting escherichia coli as shown in SEQ ID NO: 1-3; (c) an upstream primer, a downstream primer and a probe which are shown as SEQ ID NO: 4-6 and are used for detecting klebsiella pneumoniae; and (d) an upstream primer, a downstream primer and a probe which are shown as SEQ ID NO: 10-12 and are used for detecting the staphylococcus aureus. 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 their application for detecting pathogens of 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, parasites, etc., which can lead to bacteremia, septicemia and sepsis, 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 operations, the increasing use of broad-spectrum antibacterial drugs and corticosteroid drugs, the incidence and mortality of bloodstream infection have been increasing year by year. After the occurrence of bloodstream infection, the prognosis may be poor, resulting in an extended hospital stay, thus increasing the economic burden on patients. The earlier the pathogen of bloodstream infection is identified and the correct antibacterial drug treatment is selected, the mortality of patients will be reduced by about 7%. Therefore, it is crucial to quickly and timely identify the pathogenic bacteria of bloodstream infection.

[0004] For a long time, there have been many problems in the clinical diagnosis and treatment of BSI, such as early rapid diagnosis and precise treatment, which urgently need to be continuously improved. 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 and false positive rate; (2) MALD-TOF MS technology, which has good identification efficiency for Gram-negative bacteria (>90% in line with subsequent culture results), but still needs to be cautious when used to identify Gram-positive bacteria (about 80% in line with subsequent culture results); (3) Next-Generation Sequencing (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 use PCR to enrich the DNA of the detected sample, so there will be amplification preference for some segments, resulting in missed detection and insufficient accuracy.

[0005] Therefore, it is necessary 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, there is provided a primer-probe combination (or primer-probe composition) for detecting pathogens of bloodstream infection, and the primer-probe combination (or primer-probe composition) includes: (a) Forward and reverse primers and a probe for detecting coagulase-negative Staphylococcus 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 Escherichia coli as shown in SEQ ID NOs: 1-3; (c) Forward and reverse primers and a probe for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 4-6; (d) Forward and reverse primers and a probe for detecting Staphylococcus aureus as shown in SEQ ID NOs: 10-12.

[0009] (e) A combination of any two or more of the above (b)-(d).

[0010] In a preferred aspect, the present invention provides a primer-probe combination for detecting bloodstream infection pathogens, the primer-probe combination comprising: (a) Forward and reverse primers and a probe for detecting coagulase-negative Staphylococcus as shown in SEQ ID NOs: 7-9; (b) Forward and reverse primers and a probe for detecting Escherichia coli as shown in SEQ ID NOs: 1-3; (c) Forward and reverse primers and a probe for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 4-6; and (d) Forward and reverse primers and a probe for detecting Staphylococcus aureus as shown in SEQ ID NOs: 10-12.

[0011] In another preferred embodiment, the primer-probe combination further comprises primers and a probe for detecting other bloodstream 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 as shown in SEQ ID NOs: 13-15.

[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 another preferred example, the coagulase-negative staphylococci include: Staphylococcus hominis, Staphylococcus capitis, Staphylococcus simulans, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus caprae, Staphylococcus lugdunensis, Staphylococcus warneri, Staphylococcus xylosus, and Staphylococcus sciuri.

[0016] In another preferred example, the fluorescent groups of the probes in the combination are different from each other and do not interfere with each other.

[0017] As used herein, "different from each other and do not interfere with each other" 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. These groups have absorbance values that are not close to each other, and different channels can be selected, so they do not interfere with each other.

[0018] In another preferred example, the fluorescent reporter group of the coagulase-negative staphylococci probe is ROX; the fluorescent reporter group of the Escherichia coli probe is FAM; the fluorescent reporter group of the Klebsiella pneumoniae probe is HEX; the fluorescent reporter group of the Staphylococcus aureus probe is CY5.

[0019] In another preferred example, the fluorescent reporter group of the probe for detecting the internal reference is Q705.

[0020] In another preferred example, the 3'-end of the probe further has a fluorescent quenching group.

[0021] In another preferred example, the fluorescent quenching group is selected from the group consisting of: BHQ1, BHQ2, BHQ3.

[0022] In another preferred example, the quenching group at the 3'-end of the Escherichia coli probe and the Klebsiella pneumoniae probe is BHQ1; the quenching group at the 3'-end of the coagulase-negative staphylococci probe and the Staphylococcus aureus probe is BHQ2.

[0023] In another preferred example, the quenching group at the 3'-end of the internal reference is BHQ3.

[0024] In another preferred example, the components of the primer-probe combination are each present in a separate package.

[0025] In another preferred example, the components of the primer-probe combination are present in the same package.

[0026] In another preferred example, the components of the combination are present in a mixed form.

[0027] In another preferred example, the upstream and downstream primers and probe for detecting coagulase-negative Staphylococcus are designed based on specific conserved regions of the sodA gene of coagulase-negative Staphylococcus.

[0028] In another preferred example, the upstream, downstream primers and probe for detecting Escherichia coli are designed based on specific conserved regions of the ydhQ gene of Escherichia coli.

[0029] In another preferred example, the upstream and downstream primers and probe for detecting Klebsiella pneumoniae are designed based on specific conserved regions of the phoE gene of Klebsiella pneumoniae.

[0030] In another preferred example, the upstream and downstream primers and probe for detecting Staphylococcus aureus are designed based on specific conserved regions of the BioY gene of Staphylococcus aureus.

[0031] In another preferred example, a fluorescent reporter gene is linked to the 5'-end of the probe sequence, and a fluorescent quenching group is linked to the 3'-end.

[0032] 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: coagulase-negative Staphylococcus, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus.

[0033] In another preferred example, the coagulase-negative Staphylococcus includes: Staphylococcus hominis, Staphylococcus capitis, Staphylococcus simulans, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus caprae, Staphylococcus lugdunensis, Staphylococcus warneri, Staphylococcus xylosus and Staphylococcus sciuri.

[0034] In the third aspect of the present invention, there is provided a kit for detecting blood-stream infection pathogens, which kit includes the primer-probe combination as described in the first aspect of the present invention.

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

[0036] In another preferred example, 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 example, the negative control product is normal human leukocyte genomic DNA (tgDNA).

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

[0039] In another preferred example, the positive control product is at least one of fragment plasmids or fragment DNAs of coagulase-negative staphylococcus, escherichia coli, klebsiella pneumoniae, and staphylococcus aureus.

[0040] In another preferred example, the positive control product is a mixture of the genomes of coagulase-negative staphylococcus, escherichia coli, klebsiella pneumoniae, and staphylococcus aureus.

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

[0042] In another preferred example, the primer-probe combination further includes: (e) upstream and downstream primers and a probe for detecting an internal reference as shown in SEQ ID NOs: 13-15.

[0043] In another preferred example, the primers and probe for detecting the internal reference are designed for a specific conserved region of the human EGFR gene.

[0044] In another preferred example, the kit further includes a PCR reaction solution.

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

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

[0047] In another preferred example, the kit further includes an instruction manual, and the instruction manual indicates that the kit is used for detecting blood-stream infection pathogens.

[0048] In another preferred example, the kit further includes a blank control, and the blank control is ultrapure water.

[0049] In the 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.

[0050] In another preferred example, the detection includes the following steps: (i) Extracting free DNA from a sample to be tested; (ii) Performing digital 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) Read the fluorescence data and calculate the copy concentration of the target sequence to obtain the detection result.

[0051] 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 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 no detection; (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 droplets after re - examination, it is determined as a negative result, and report no detection.

[0052] In another preferred embodiment, the test sample is selected from the group consisting of: whole blood, plasma, serum, or a combination thereof.

[0053] In the fifth aspect of the present invention, a method for detecting blood - stream - infecting pathogens is provided, comprising the following steps: (i) Extract the free DNA from the test sample; (ii) Perform 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; (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 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 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 no detection; (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 droplets after re - examination, it is determined as a negative result, and report no detection.

[0055] In another preferred embodiment, the test sample is selected from the group consisting of: whole blood, plasma, serum, or a combination thereof.

[0056] In another preferred embodiment, the method is non - diagnostic and non - therapeutic.

[0057] In another preferred embodiment, the method is an in vitro method.

[0058] 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 embodiments) 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

[0059] Figure 1 The figure shows the detection result diagram of Escherichia coli positive detected by using the primer-probe combination of the present invention.

[0060] Figure 2 The figure shows the detection result diagram of Klebsiella pneumoniae positive detected by using the primer-probe combination of the present invention.

[0061] Figure 3 The figure shows the detection result diagram of Staphylococcus aureus positive detected by using the primer-probe combination of the present invention.

[0062] Figure 4 The figure shows the detection result diagram of coagulase-negative Staphylococcus positive detected by using the primer-probe combination of the present invention.

[0063] Figure 5 The figure shows the detection result diagram of internal reference (EGFR) positive detected by using the primer-probe combination of the present invention.

[0064] Figure 6 The figure shows the concentration result of amplifying the target gene of Escherichia coli by using the multiplex PCR combination.

[0065] Figure 7 The figure shows the concentration result of single amplification of the target gene of Escherichia coli.

[0066] Figure 8 The figure shows the concentration result of amplifying the target gene of Klebsiella pneumoniae by using the multiplex PCR combination.

[0067] Figure 9 The figure shows the concentration result of single amplification of the target gene of Klebsiella pneumoniae.

[0068] Figure 10 The figure shows the concentration result of amplifying the target gene of coagulase-negative Staphylococcus by using the multiplex PCR combination.

[0069] Figure 11 The figure shows the concentration result of single amplification of the target gene of coagulase-negative Staphylococcus.

[0070] Figure 12 The figure shows the concentration result of amplifying the target gene of Staphylococcus aureus by using the multiplex PCR combination.

[0071] Figure 13Shows the concentration results of single amplification of the target gene of Staphylococcus aureus.

[0072] Figure 14 Shows the detection sensitivity of Escherichia coli in Example 2.

[0073] Figure 15 Shows the detection sensitivity of Klebsiella pneumoniae in Example 2.

[0074] Figure 16 Shows the detection sensitivity of coagulase-negative Staphylococcus (Staphylococcus hominis) in Example 2.

[0075] Figure 17 Shows the detection sensitivity of coagulase-negative Staphylococcus (Staphylococcus capitis) in Example 2.

[0076] Figure 18 Shows the detection sensitivity of coagulase-negative Staphylococcus (Staphylococcus simulans) in Example 2.

[0077] Figure 19 Shows the detection sensitivity of coagulase-negative Staphylococcus (Staphylococcus saprophyticus) in Example 2.

[0078] Figure 20 Shows the detection sensitivity of coagulase-negative Staphylococcus (Staphylococcus epidermidis) in Example 2.

[0079] Figure 21 Shows the detection sensitivity of coagulase-negative Staphylococcus (Staphylococcus haemolyticus) in Example 2.

[0080] Figure 22 Shows the detection sensitivity of coagulase-negative Staphylococcus (Staphylococcus caprae) in Example 2.

[0081] Figure 23 Shows the detection sensitivity of coagulase-negative Staphylococcus (Staphylococcus lugdunensis) in Example 2.

[0082] Figure 24 Shows the detection sensitivity of coagulase-negative Staphylococcus (Staphylococcus warneri) in Example 2.

[0083] Figure 25 Shows the detection sensitivity of coagulase-negative Staphylococcus (Staphylococcus xylosus) in Example 2.

[0084] Figure 26 Shows the detection sensitivity of coagulase-negative Staphylococcus (Staphylococcus sciuri) in Example 2.

[0085] Figure 27 Shows the detection sensitivity of Staphylococcus aureus in Example 2.

[0086] Figure 28 Shows the specific detection results of the mixed pathogens in Example 3. Detailed implementation mode

[0087] 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: coagulase-negative staphylococci, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Experiments of the present invention show that there is no difference in the amplification concentration results between singleplex and multiplex PCR, so there is no mutual influence among the primers and probes of the present invention that causes a decrease in the PCR amplification efficiency. Moreover, the minimum detection sensitivity of the digital PCR method of the present invention is 5 copies / reaction, and the specificity is good. On this basis, the present invention was completed.

[0088] 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.

[0089] As used herein, the term "comprising" or "including" can be open-ended, semi-closed, and closed. In other words, the term also includes "consisting essentially of..." or "consisting of...".

[0090] 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 stated range and, where appropriate, fractional values thereof (such as one-tenth and one-hundredth of an integer).

[0091] 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.

[0092] 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 rate, high mortality rate, high treatment cost, and difficult diagnosis.

[0093] Currently, blood culture is the gold standard for the 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 LμMinex. 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 the registration certificate from the National Medical Products Administration (NMPA).

[0094] The pathogen laboratory detection of BSI takes a long time and is difficult to diagnose, which is a major challenge that urgently needs to be solved in current clinical emergency departments and infection departments and seriously 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.

[0095] The multiplex digital PCR (dPCR) rapid detection method randomly divides the fluorescence quantitative reaction system into hundreds 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 the highly difficult detection of BSI with low levels of pathogenic microorganisms, complex blood components, and high human-derived 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 the low detection rate and long reporting cycle of current blood stream infection pathogens.

[0096] Escherichia coli, Klebsiella pneumoniae, coagulase-negative staphylococci, and Staphylococcus aureus Escherichia coli is a common pathogen of blood stream infection, especially secondary blood stream infection caused by urinary tract infection. The endotoxin it produces may lead to sepsis and septic shock.

[0097] Klebsiella pneumoniae is an important pathogen of hospital-acquired blood stream infection, especially multi-drug resistant strains (such as Klebsiella pneumoniae producing carbapenemase), which can cause severe sepsis and high mortality.

[0098] Coagulase-negative staphylococci (CoNS) are the main pathogens of catheter-related bloodstream infections, especially in patients with low immunity or long-term catheter use. Although they have low virulence, they are prone to form biofilms and are difficult to remove. It includes Staphylococcus hominis, Staphylococcus capitis, Staphylococcus simulans, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus caprae, Staphylococcus lugdunensis, Staphylococcus warneri, Staphylococcus xylosus, and Staphylococcus sciuri, etc.

[0099] Staphylococcus aureus is the main pathogen of bloodstream infections, especially methicillin-resistant Staphylococcus aureus (MRSA), which can cause serious complications such as sepsis and endocarditis, and has a relatively high mortality rate.

[0100] In summary, coagulase-negative staphylococci and Staphylococcus aureus are Gram-positive bacteria. The former is more common in catheter-related infections, while the latter is associated with severe bloodstream infections, especially MRSA. Escherichia coli and Klebsiella pneumoniae are Gram-negative bacteria, which are common in hospital-acquired bloodstream infections and are prone to cause sepsis.

[0101] The detection method of the present invention The present invention provides a method for detecting pathogens causing bloodstream infections, comprising the following steps: 1) According to the gene sequences of the pathogens, specific primer-probes (including internal reference primers and probes) are designed. The pathogens include Escherichia coli, Klebsiella pneumoniae, coagulase-negative staphylococci, and Staphylococcus aureus. For coagulase-negative staphylococci, degenerate primer-probes are designed, which can detect coagulase-negative staphylococci more comprehensively and avoid missed detection. The specific primer-probe sequences are shown in Table 1 below.

[0102] Table 1

[0103] Note: W represents A / T degenerate base, and Y represents C / T degenerate base.

[0104] Among them, the 5' end of the probe sequence is also connected with a fluorescent reporter gene, and the 3' end is also connected with 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, and BHQ3.

[0105] Digital PCR detection is carried out using the above primers and probes, and the optimal detection conditions of the PCR system are optimized.

[0106] 2) Extract nucleic acids from the sample. Select a method suitable for the sample type to extract nucleic acids, where the sample types include blood cultures, peripheral blood, and peripheral blood plasma / serum. In particular, for the extraction of cell-free nucleic acids from blood samples, the steps are as follows: First, selection of blood collection tubes. If ordinary blood collection tubes are used, they should not contain heparin, and plasma needs to be separated within 4 hours. If the blood cannot be processed in time, blood collection tubes with cell stabilization technology should be selected.

[0107] Second, separation of plasma. Separation of plasma: In the first step, centrifuge at low speed to remove cells at 4°C, 1600g, for 10 minutes.

[0108] Third, extraction. Use a special extraction kit for plasma cell-free nucleic acids and perform extraction according to the instructions.

[0109] 3) Digital PCR.

[0110] First, preparation of the PCR system. Prepare the PCR system in the reagent preparation area as follows: 5×HS Taq buffer, containing Mg 2+ 6 μL, dNTPs (10 mM each) 0.75 μL, hot-start Taq DNA 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.

[0111] Preferably, the final concentration of each primer is 0.33 μM, and the final concentration of each probe is 0.17 μM.

[0112] Second, addition of the template. Add the template in the following order in the sample preparation area: the sample to be tested, blank control, negative control, positive control. The negative control is normal human leukocyte genomic DNA (abbreviated as "tgDNA") with a copy concentration of 1000 copies / μL, and the positive control solution has 50 copies / μL of all target sites. The blank control is ultrapure water.

[0113] Third, generation of droplets. Generate droplets according to the instrument requirements.

[0114] Fourth, PCR. Through the annealing temperature optimization experiment, it was found that the digital PCR effect was optimal when annealing at 56°C for 15 s. Perform PCR according to the following procedure: 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.

[0115] Fifth, scanning. Judge whether the sample is positive or negative according to the scanning results.

[0116] The determination of the detection results 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 no detection; (3) If there is 1 or 2 positive droplets, it indicates a gray area, and re-examination is recommended; if there are still < 3 positive droplets after re-examination, it is determined as a negative result, and report no detection.

[0117] The kit of the present invention The present invention provides a kit for detecting Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococcus, and Staphylococcus aureus. The kit includes PCR primer probes (including primer probes for internal reference), reaction system, reaction conditions, negative control DNA sample, and positive control DNA sample. The following content: Primers and probes: Synthesize the oligonucleotide fragments and modified oligonucleotide fragments shown in Table 1, and dissolve them in TE (10 μM), as described in the previous part.

[0118] The reaction conditions are as described in the previous part.

[0119] PCR reagent: Use 5×HS Taq Buffer with Mg for DNA polymerase 2+ The reaction system is as described in the previous part.

[0120] Control: The blank control is water, the negative control is normal human leukocyte genome (tgDNA), and the copy concentration is 1000 copies / μL. The positive control is a mixture of the genomes 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.

[0121] The main advantages of the present invention include: (a) Compared with the prior art such as blood culture, MALD-TOF MS, NGS, etc., the present invention uses Taqman probes in combination with digital PCR method, which can solve problems such as low sensitivity, poor specificity, high requirements for sample type and quality, and complex positive interpretation methods.

[0122] (b) High sensitivity: Since this method uses a digital PCR platform, it can divide the reaction system into about 20,000 tiny reactions. In theory, it can detect single-copy mutations and has 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.

[0123] (c) Strong specificity: Specific primers and probes are designed respectively for the specific sequences of Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococcus, and Staphylococcus aureus, which can effectively amplify the target position without amplifying other sequences.

[0124] (d) Loose requirements for sample type and quality, and strong anti-interference ability. Due to the high sensitivity of the present invention, the applicable sample type is peripheral blood sample (this sample is relatively easy to obtain, but has low DNA content and is fragmented); moreover, due to the uniqueness of its digital PCR platform, that is, it can divide the reaction system into about 20,000 small systems, and at the same time can also divide the interfering substances into about 20,000 parts, which can greatly reduce the influence of interfering substances on the reaction. Of course, it can also detect samples with a more complex background. This cannot be achieved by other platforms.

[0125] (e) Simple positive interpretation method: Since the present invention uses an absolute quantification method, there is no need to set a control standard curve. The result can be determined whether it contains the target mutant template 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 directly report the infectious 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 no detection; (3) If there is 1 or 2 positive droplets, it indicates a gray area, and re-examination is recommended; if there are still < 3 positive micro-droplets after re-examination, it is determined as a negative result, and report no detection.

[0126] 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 stated, percentages and parts are in mole percentages and mole parts.

[0127] Example 1 Screening of Primers and Probes for Detecting Escherichia coli, Klebsiella pneumoniae, Coagulase-Negative Staphylococci, and Staphylococcus aureus by Digital PCR 1.1 Primer and Probe Design Design of primers and probes for detecting pathogens: Multiple sets of primers and probes were designed based on the specific genes of Escherichia coli, Klebsiella pneumoniae, coagulase-negative staphylococci, and Staphylococcus aureus. Finally, sequences that do not affect each other and have better amplification efficiency were screened out as shown in Table 2 below. Among them, 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.

[0128] The upstream and downstream primers and probe for detecting coagulase-negative staphylococci were designed based on specific conserved regions of the sodA gene of coagulase-negative staphylococci. The upstream, downstream, and probe for detecting Escherichia coli were designed based on specific conserved regions of the ydhQ gene of Escherichia coli. The upstream and downstream primers and probe for detecting Klebsiella pneumoniae were designed based on specific conserved regions of the phoE gene of Klebsiella pneumoniae. The upstream and downstream primers and probe for detecting Staphylococcus aureus were designed based on specific conserved regions of the BioY gene of Staphylococcus aureus.

[0129] Design of primers and probes for internal reference: Primers and probes were screened from multiple human genes, aiming to have no mutual influence with the primers and probes for detecting the above pathogens and have better 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 probe for detecting the internal reference were designed based on specific conserved regions of the human EGFR gene.

[0130] Table 2

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

[0132] 1.2 PCR System 5×HS Taq Buffer, containing Mg 2+6 μL, 0.75 μL of dNTPs (10 mM each), 0.2 μL of hot-start 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 made up to 30 μL with water.

[0133] 1.4 Chip Scanning After testing, it can effectively detect Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococcus, Staphylococcus aureus, and the internal reference ( Figures 1-5 ). In addition, nine coagulase-negative Staphylococci in common clinical samples were detected, and the detection results are shown in Table 3 below: Table 3

[0134] By comparing the single and multiplex results of each detected target, it can be seen that there is no difference in the amplification concentration results between single and multiplex (Table 4). Therefore, there is no mutual influence between primers and probes that leads to a decrease in the PCR amplification efficiency. For details, see Figures 6-13 .

[0135] Table 4

[0136] Example 2 Verification of the Sensitivity of Digital PCR for Detecting Escherichia coli, Klebsiella pneumoniae, Coagulase-Negative Staphylococcus, and Staphylococcus aureus 2.1 Experimental Method The detected strains include: Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococcus, and Staphylococcus aureus. The coagulase-negative Staphylococcus includes: Staphylococcus hominis, Staphylococcus capitis, Staphylococcus simulans, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus caprae, Staphylococcus lugdunensis, Staphylococcus warneri, Staphylococcus xylosus, and Staphylococcus sciuri.

[0137] Calculate the copy number of each pathogen template (copies / μL) according to Example 1, and dilute each pathogen template to 2 copies / μL and 1 copy / μL with TE to prepare a gradient dilution template.

[0138] Verify the digital PCR system: 5×HS Taq buffer, containing Mg 2+ 6 μL, dNTPs (each 10 mM) 0.75 μL, hot start Taq DNA 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.

[0139] Add the gradient-diluted template in the following order in the sample preparation area: 2 copies / μL and 1 copy / μL of each pathogen genome.

[0140] Generate droplets from the PCR reaction system in the same method 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.

[0141] 2.2 Experimental results The digital PCR results are shown in Figures 14-27 . In addition, the digital PCR method of the present invention can effectively detect positive points when detecting a 1 cps / μL template, that is, the lowest detection sensitivity of the digital PCR method of the present invention is 5 copies / reaction.

[0142] Specific verification of the digital PCR method for detecting Escherichia coli, Klebsiella pneumoniae, coagulase-negative Staphylococcus, and Staphylococcus aureus in Example 3 3.1 Experimental method Design primers and probes for different pathogens, calculate the copy number of each pathogen template (copies / μL) according to Example 1, and dilute each pathogen template to 10,000 copies / μL with TE. Then mix each pathogen in equal proportions in groups of 5 (a total of 8 groups or 8 samples), and the combinations are shown in Table 5 below: Table 5

[0143] Verify the digital PCR system: 5×HS Taq buffer, containing Mg 2+ 6 μL, dNTPs (10 mM each) 0.75 μL, hot start Taq DNA 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, and make up to 30 μL with water.

[0144] Add templates in the following order in the sample preparation area: blank control, negative control, and mixed pathogen template. The blank control is water, the negative control is tgDNA, and the mixed pathogen template is as shown in Table 5 above.

[0145] Generate droplets for the PCR reaction system in the same method 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.

[0146] 3.2 Experimental results The results of digital PCR are shown in Figure 28 , and for the digital PCR method of the present invention, using water or tgDNA as the template, the background is clean and there is no contamination. In addition, the digital PCR method of the present invention has no cross-reaction when detecting common clinical pathogens other than the detection target at a concentration of 2000 copies / μL, indicating good specificity.

[0147] Discussion The present invention provides a method and application for detecting blood-stream infection pathogens, including primers and probes. More specifically, the pathogens involved include coagulase-negative staphylococci, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus.

[0148] The primer-probe composition provided by the present invention mainly uses the multiplex fluorescence PCR analysis method to detect and distinguish different pathogens by designing primer-probes on specific genes of different pathogens. Among them, the 5'-end of the Escherichia coli probe sequence is provided with a FAM fluorophore, and the 3'-end is provided with a BHQ1 group; the 5'-end of the Klebsiella pneumoniae probe sequence is provided with a HEX fluorophore, and the 3'-end is provided with a BHQ1 group; the 5'-end of the coagulase-negative staphylococci probe sequence is provided with a ROX fluorophore, and the 3'-end is provided with a BHQ2 group; the 5'-end of the Staphylococcus aureus probe sequence is provided with a CY5 fluorophore, and the 3'-end is provided with a BHQ2 group.

[0149] The digital PCR detection kit for pathogens in blood of the present invention can detect nucleic acid fragments of pathogens in blood, making up for the disadvantages of low throughput and long time consumption of conventional blood culture detection methods, and providing comprehensive, accurate, and low-cost etiological diagnosis for clinical practice in a timely manner, providing an important reference for individualized medication and precision medicine.

[0150] All documents mentioned in the present invention are cited in this application as references, just as if each document is cited separately as a 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 this 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 coagulase-negative staphylococci as shown in SEQ ID NOs: 7-9; (b) upstream and downstream primers and a probe for detecting Escherichia coli as shown in SEQ ID NOs: 1-3; (c) upstream and downstream primers and a probe for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 4-6; and (d) upstream and downstream primers and a probe for detecting Staphylococcus aureus as shown in SEQ ID NOs: 10-12.

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

3. 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 an internal reference gene as shown in SEQ ID NOs: 13-15.

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

5. 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: coagulase-negative staphylococci, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus.

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, wherein The kit further includes a negative control product and a positive control product.

8. The kit according to claim 7, characterized in that, The positive control product is a mixture of genomes of coagulase-negative staphylococci, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus; the negative control product is normal human leukocyte genomic DNA (tgDNA).

9. Use of the primer-probe combination according to claim 1, 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 digital PCR on the free DNA obtained in step (i) using the primer-probe combination as described in claim 1; (iii) Reading the fluorescence data and calculating the copy concentration of the target sequence, so as to obtain the detection result.

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