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

Abstract

The present application provides a method and a kit for detecting blood stream infection pathogens and application. Specifically, the present application provides a primer probe combination for detecting blood stream infection pathogens, which comprises: (a) upper and lower primers and probes for detecting Candida as shown in SEQ ID NO: 4~6; (b) upper and lower primers and probes for detecting Candida glabrata as shown in SEQ ID NO: 1~3; (c) upper and lower primers and probes for detecting Candida tropicalis as shown in SEQ ID NO: 7~9; and (d) upper and lower primers and probes for detecting Cryptococcus as shown in SEQ ID NO: 10~12. The primer probe combination of the present application can be used to achieve rapid, high sensitivity and high specificity detection of blood stream infection pathogens.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular diagnosis, in particular, to a method and kit for detecting blood stream infection pathogens and application. BACKGROUND

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

[0003] Due to factors such as increased invasive procedures, increased use of broad-spectrum antibacterial drugs and corticosteroid drugs, the incidence and mortality of bloodstream infection have increased year by year. After bloodstream infection occurs, the prognosis may be poor, leading to prolonged hospitalization time and thus increasing the economic burden of patients. The earlier the pathogen of bloodstream infection is identified, the more correct the antibacterial drug treatment is, and the mortality of patients decreases by about 7%. Therefore, it is crucial to identify the pathogenic bacteria of bloodstream infection in a timely and rapid manner.

[0004] For a long time, there are many problems in the diagnosis and treatment of clinical BSI, such as early rapid diagnosis, precise treatment, etc., which need to be continuously improved. The current detection methods of BSI mainly include: (1) blood culture, but it has the disadvantages of low detection rate, long detection time, and high contamination and false positive rate; (2) MALD-TOF MS technology, this technology has good identification efficiency (>90% consistent with subsequent culture results) for gram-negative bacteria, but it still needs to be cautious when used for identifying gram-positive bacteria (about 80% consistent with subsequent culture results); (3) Next-Generation Sequencing (NGS), the detection cost is high, so it is not suitable for rapid detection required by general bloodstream infection patients, and NGS needs to use PCR method to enrich the DNA of the sample to be detected, so there is amplification bias for some sections, which leads to the occurrence of missed detection and the problem of insufficient accuracy.

[0005] Therefore, it is necessary to develop a method and kit for detecting bloodstream infection with rapidity, high specificity, high sensitivity, and strong anti-interference ability. SUMMARY

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

[0007] In a first aspect of the present application, a primer probe combination (or primer probe composition) for detecting blood stream infection pathogens is provided, which comprises:

[0008] (a) the upstream and downstream primers and probe for detecting Candida as set forth in SEQ ID NOs: 4-6;

[0009] (b) the upstream and downstream primers and probe for detecting C. glabrata as set forth in SEQ ID NOs: 1-3;

[0010] (c) the upstream and downstream primers and probe for detecting C. tropicalis as set forth in SEQ ID NOs: 7-9; and

[0011] (d) the upstream and downstream primers and probe for detecting Cryptococcus as set forth in SEQ ID NOs: 10-12.

[0012] In another preferred embodiment, the primer probe combination further comprises primers and probes for detecting other blood stream infection pathogens.

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

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

[0015] (e) the upstream and downstream primers and probe for detecting an internal reference (or internal reference gene) as set forth in SEQ ID NOs: 17-19.

[0016] In another preferred embodiment, the primers and probes for detecting an internal reference are designed for a specific conserved region of the human EGFR gene.

[0017] In another preferred embodiment, the Candida comprises C. albicans, C. glabrata, C. tropicalis, C. pseudotropicalis, C. dubliniensis, C. guilliermondii, C. parapsilosis, C. kefyr, and C. auris.

[0018] In another preferred embodiment, the Cryptococcus comprises C. neoformans and C. gattii.

[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 from each other and do not interfere with each other" means that the fluorescent groups used for each probe in the composition are different from each other and do not affect the detection of each other, i.e., different channels can be used for detection. For example, FAM, HEX, ROX, CY5, and Q705 can be used, which have different absorbance values and can be selected for different channels, and thus do not interfere with each other.

[0021] In another preferred embodiment, the fluorescent reporter group of the Candida probe is HEX; the fluorescent reporter group of the C. glabrata probe is FAM; the fluorescent reporter group of the C. tropicalis probe is ROX; and the fluorescent reporter group of the Cryptococcus probe is CY5.

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

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

[0024] In another preferred embodiment, the fluorescent quenching group is selected from the group consisting of BHQ1, BHQ2, and BHQ3.

[0025] In another preferred embodiment, the quenching group at the 3' end of the C. glabrata and Candida probes is BHQ1; the quenching group at the 3' end of the C. tropicalis and Cryptococcus probes is BHQ2.

[0026] In another preferred embodiment, the quenching group at the 3' end of the internal control 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, each component of the primer probe combination is present in the same package.

[0029] In another preferred embodiment, each component of the combination is present in a mixed form.

[0030] In another preferred embodiment, 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.

[0031] In a second aspect of the present application, there is provided use of the primer probe combination as described in the first aspect of the present application, for preparing a kit for detecting a blood stream infection pathogen; wherein the pathogen includes Candida, C. glabrata, C. tropicalis, and Cryptococcus.

[0032] In another preferred embodiment, the Candida includes C. albicans, C. glabrata, C. tropicalis, C. pseudotropicalis, C. dubliniensis, C. guilliermondii, C. parapsilosis, C. kefyr, and C. auris.

[0033] In another preferred embodiment, the Cryptococcus includes C. neoformans and C. gattii.

[0034] In a third aspect of the present application, there is provided a kit for detecting a blood stream infection pathogen, the kit comprising the primer probe combination as described in the first aspect of the present application.

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

[0036] In another preferred embodiment, the negative quality control is at least one of DEPC-H2O, normal saline, an internal standard gene pseudovirus, and normal human leukocyte genomic DNA.

[0037] In another preferred embodiment, the negative quality control 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 is at least one of a fragment plasmid or a fragment DNA of Candida, C. glabrata, C. tropicalis, and Cryptococcus.

[0040] In another preferred embodiment, the positive quality control is a mixture of genomes of Candida, C. glabrata, C. tropicalis, and Cryptococcus.

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

[0042] In another preferred embodiment, the primer-probe combination further comprises:

[0043] (e) an upstream primer and a probe for detecting the internal reference as shown in SEQ ID NOs: 17-19.

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

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

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

[0047] In another preferred embodiment, the kit further comprises an instruction manual, which indicates that the kit is used for detecting blood stream 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 application, there is provided use of the primer probe combination according to the first aspect of the present application or the kit according to the third aspect of the present application, for the preparation of a reagent for detecting a blood stream infectious pathogen.

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

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

[0052] (ii) performing digital PCR on the free DNA obtained in step (i) using the primer probe combination according to the first aspect of the present application or the kit according to the third aspect of the present application;

[0053] (iii) reading the fluorescence data and calculating the copy concentration of the sequence of interest, thereby obtaining a detection result.

[0054] In another preferred embodiment, in step (iii), the detection result is determined as follows:

[0055] (1) if the positive droplets are ≥ 3 and the clustering is consistent with the positive control, it is suggested to be a positive result, and the infectious pathogen and the copy number are directly reported;

[0056] (2) if there are no positive droplets and the clustering is consistent with the negative control, it is suggested to be a negative result, and it is reported that nothing is detected;

[0057] (3) if there are 1 or 2 positive droplets, it is suggested to be a gray area, and retesting is recommended; if the retesting is still < 3 positive droplets, it is determined to be a negative result, and it is reported that nothing is 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 application, there is provided a method for detecting a blood stream infectious pathogen, comprising the following steps:

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

[0061] (ii) performing digital PCR on the free DNA obtained in step (i) using the primer probe combination according to the first aspect of the present application or the kit according to the third aspect of the present application;

[0062] (iii) reading the fluorescence data and calculating the copy concentration of the sequence of interest, thereby obtaining a detection result.

[0063] In another preferred embodiment, in step (iii), the detection result is determined as follows:

[0064] (1) If the positive droplets are ≥3, and the clustering and positive control are consistent, it is suggested that the result is positive, and the infection pathogen and copy number are directly reported;

[0065] (2) If there are no positive droplets, and the clustering and negative control are consistent, it is suggested that the result is negative, and it is reported that no pathogen is detected;

[0066] (3) If there are 1 or 2 positive droplets, it is suggested that the result is in the gray zone, and retesting is recommended; if the retesting is still <3 positive droplets, it is determined that the result is negative, and it is reported that no pathogen is 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 application, each of the technical features described above and each of the technical features described in detail below (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 A detection result chart showing a positive result of Candida albicans detected using the multiplex primer probe combination of the present application.

[0072] Figure 2 A detection result chart showing a positive result of Candida (Candida albicans) detected using the multiplex primer probe combination of the present application.

[0073] Figure 3 A detection result chart showing a positive result of Candida tropicalis detected using the multiplex primer probe combination of the present application.

[0074] Figure 4 A detection result chart showing a positive result of Cryptococcus (Cryptococcus neoformans) detected using the multiplex primer probe combination of the present application.

[0075] Figure 5 A detection result chart showing a positive result of an internal reference detected using the multiplex primer probe combination of the present application.

[0076] Figure 6 A concentration result chart showing amplification of the target gene of Candida albicans using the multiplex primer probe combination.

[0077] Figure 7 A concentration result chart showing single amplification of the target gene of Candida albicans.

[0078] Figure 8 Concentration results showing amplification of Candida (C. albicans) target genes using multiplex primer probe combinations.

[0079] Figure 9 Concentration results showing singleplex amplification of Candida (C. albicans) target genes.

[0080] Figure 10 Concentration results showing amplification of Candida (C. tropicalis) target genes using multiplex primer probe combinations.

[0081] Figure 11 Concentration results showing singleplex amplification of Candida (C. tropicalis) target genes.

[0082] Figure 12 Concentration results showing amplification of Cryptococcus (C. neoformans) target genes using multiplex primer probe combinations.

[0083] Figure 13 Concentration results showing singleplex amplification of Cryptococcus (C. neoformans) target genes.

[0084] Figure 14 Detection sensitivity of C. glabrata in Example 2 is shown.

[0085] Figure 15 Detection sensitivity of Candida (C. pseudotropicalis) in Example 2 is shown.

[0086] Figure 16 Detection sensitivity of Candida (C. dubliniensis) in Example 2 is shown.

[0087] Figure 17 Detection sensitivity of Candida (C. guilliermondii) in Example 2 is shown.

[0088] Figure 18 Detection sensitivity of Candida (C. parapsilosis) in Example 2 is shown.

[0089] Figure 19 Detection sensitivity of Candida (C. kefyr) in Example 2 is shown.

[0090] Figure 20 Detection sensitivity of Candida (C. auris) in Example 2 is shown.

[0091] Figure 21 Detection sensitivity of C. tropicalis in Example 2 is shown.

[0092] Figure 22The detection sensitivity of Cryptococcus (C. neoformans) in Example 2 is shown.

[0093] Figure 23 The detection sensitivity of Cryptococcus (C. gattii) in Example 2 is shown.

[0094] Figure 24 The specific detection results of mixed pathogens in Example 3 are shown.

[0095] OVERALL DESCRIPTION OF DRAWINGS: The gray markings present in the drawings represent a supplemental explanation of the figure, used to hint at the specific single channel fluorescence information of the individual figure below it, i.e. the signal intensity measured in a certain specific fluorescence detection channel (e.g. FAM, HEX, ROX, CY5) of the droplet. DETAILED DESCRIPTION

[0096] The inventors have unexpectedly developed primers and probes that can be used to rapidly and highly specifically and sensitively detect blood stream infection pathogens, including Candida, C. glabrata, C. tropicalis, and Cryptococcus, through extensive and in-depth research and a large amount of screening. The experiments of the present application show that there is no difference in the amplification concentration results of single and multiplex PCR, and thus there is no mutual influence between the primers and probes of the present application that causes a decrease in PCR amplification efficiency. Also, the digital PCR method of the present application has a minimum detection sensitivity of 5 copies / reaction, and good specificity. On this basis, the present application is completed.

[0097] TERMS

[0098] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, unless specifically stated otherwise, each of the following terms has the meaning given below. Additional definitions are set forth throughout the application.

[0099] As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0100] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood to include the values expressly indicated within the given range, as well as values which are substantially the same as those indicated within the given range (e.g., one tenth and one hundredth of an integer within a given range).

[0101] As used herein, the term "and / or" relates to and encompasses any and all possible combinations of one or more of the associated listed items.

[0102] Blood stream infection

[0103] Bloodstream infection (BSI) is an infection caused by pathogenic microorganisms entering the blood circulation. The main pathogens include bacteria, fungi and viruses. BSI has the characteristics of high incidence, high mortality, high treatment cost and difficult diagnosis.

[0104] BSI has a 40% probability of developing into sepsis. In China, there are 6.12 million new cases of sepsis each year, with a 90-day mortality rate of 33.5%, an average ICU hospitalization time of 32 days, and an average treatment cost of 300,000 yuan. Research has found that for patients with septic shock, the survival rate decreases by 7.6% for every hour of delayed treatment after hypotension occurs, and the median survival rate is 42% after a 6-hour delay.

[0105] 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 by Merieux, Verigene Gram+ BC and Verigene Gram- BC by LuMinex, but these molecular detection methods are based on positive blood culture for detection. There are also some detection methods based on mNGS in China, but mNGS detection time is 1-2 days, and the cost is high, the operation is complex, and professional bioinformatics analysis personnel are needed, which is difficult to widely carry out in medical institutions.

[0106] BSI pathogen laboratory detection takes a long time, and diagnosis is difficult, which is a major challenge that needs to be addressed urgently in clinical emergency and infectious disease departments, and is a major challenge to people's life and health. Therefore, a sensitive and fast blood stream infection pathogen detection method will help to quickly identify the cause, reduce mortality, effectively improve the timeliness of diagnosis and treatment, improve medical level and efficiency.

[0107] Multiple digital PCR (dPCR) rapid detection method, by separating the fluorescent quantitative reaction system into tens of thousands of independent micro reaction units at random, and according to the Poisson distribution and the proportion of positive micro droplets to realize the absolute quantification of nucleic acid molecules. As the third generation of PCR technology, dPCR has higher sensitivity than fluorescent quantitative PCR, and has strong anti-interference ability, can realize absolute quantification, and is suitable for BSI which has low pathogenic microorganism load, complex blood components and high human DNA signal. The innovative rapid blood stream infection pathogen detection technology based on multiple digital PCR has a detection limit of 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 world's problems of low detection rate of blood stream infection pathogens and long reporting cycle.

[0108] Candida, Candida glabrata, Candida tropicalis and Cryptococcus

[0109] Candida is a type of yeast-like fungus belonging to the Ascomycota phylum. It includes various pathogenic and non-pathogenic species, with Candida albicans being the most common. Both C. glabrata and C. tropicalis belong to the Candida genus. Candida is one of the main pathogens of hospital-acquired bloodstream infections.

[0110] C. glabrata is one of the common pathogens of hospital-acquired bloodstream infections (candidemia), often found in immunocompromised patients (such as cancer patients, organ transplant recipients) or those who have long-term use of broad-spectrum antibiotics or central venous catheterization.

[0111] C. tropicalis is one of the common pathogens of candidemia, with strong virulence, often found in patients with hematological malignancies. Infection can lead to severe systemic infections such as sepsis and multiple organ failure.

[0112] Cryptococcus is a type of yeast-like fungus belonging to the Basidiomycota phylum. The most common pathogenic species in the Cryptococcus genus are C. neoformans and C. gattii. Cryptococcus can spread through the bloodstream, causing cryptococcosis, and further leading to central nervous system infections such as cryptococcal meningitis. Cryptococcosis has a high mortality rate in immunocompromised patients.

[0113] The detection method of the present application

[0114] The present application provides a method for detecting pathogens causing bloodstream infections, comprising the following steps:

[0115] 1) According to the genetic sequence of the pathogen, design specific primers and probes (including primers and probes for internal reference). The pathogen includes C. glabrata, Candida, C. tropicalis, and Cryptococcus. Perform digital PCR detection using the above primers and probes, and optimize the optimal detection conditions of the PCR system. Apply this method to detect patient blood samples. The specific primer and probe sequences are shown in Table 1 below.

[0116] Table 1

[0117]

[0118] Note: S represents G / C degenerate base.

[0119] Among them, the 5' end of the probe sequence is also connected with a fluorescence reporter gene, and the 3' end is also connected with a fluorescence quenching group. The fluorescence reporter group is selected from the group consisting of FAM, HEX, ROX, CY5, and Q705; the fluorescence quenching group is selected from the group consisting of BHQ1, BHQ2, and BHQ3.

[0120] 2) Extraction of nucleic acid from sample. Extract nucleic acid by a method suitable for the sample type, including blood culture, peripheral blood, peripheral blood plasma / serum. In particular, for free nucleic acid extraction of blood sample, the steps are as follows:

[0121] First, selection of blood collection tube. If a common blood collection tube is used, it cannot contain heparin, and blood plasma needs to be separated within 4h. If blood cannot be processed in time, a blood collection tube containing blood cell stabilization technology needs to be selected.

[0122] Second, separation of blood plasma. Separation of blood plasma: first step, low-speed centrifugation to remove cells, 4°C, 1600g, 10min.

[0123] Third, extraction. Use a special extraction kit for blood plasma free nucleic acid, and extract according to the requirements of the instruction manual.

[0124] 3) Digital PCR.

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

[0126] 5x 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 (10μM) 1μL, SEQ ID NO: 6 (10μM) 0.5μL, SEQ ID NO: 7 (10μM) 1μL, SEQ ID NO: 8 (10μM) 1μL, SEQ ID NO: 9 (10μM) 0.5μL, SEQ ID NO: 10 (10μM) 1μL, SEQ ID NO: 11 (10μM) 1μL, SEQ ID NO: 12 (10μM) 0.5μL, template 5μL, and water to 30μL.

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

[0128] Second, the template is added. In the sample preparation area, the template is added in the following order: the sample to be tested, the blank control, the negative control, and the positive control. The negative control is normal human white blood cell genomic DNA (referred to as "tgDNA"), with a copy concentration of 1000 copies / μL, and the positive control solution has a copy number of 50 copies / μL at all target sites. The blank control is ultrapure water.

[0129] Third, droplet generation. The droplet generation is performed according to the requirements of the instrument.

[0130] Fourth, PCR. After annealing temperature optimization experiments, it is found that the digital PCR effect is optimal at 56℃ for 15s. The PCR is performed according to the following procedure: 95℃ for 10min, 40 cycles (95℃ for 30s, 56℃ for 15s, and 72℃ for 15s), with a temperature rising and falling rate of 2℃ / s.

[0131] Fifth, scanning. According to the scanning results, it is determined whether the sample is positive or negative.

[0132] The determination of the detection results is as follows:

[0133] (1) If the positive droplets are ≥3 and the clustering is consistent with the positive control, it is indicated that the result is positive, and the infection pathogen and the copy number are directly reported.

[0134] (2) If there are no positive droplets and the clustering is consistent with the negative control, it is indicated that the result is negative, and it is reported that nothing is detected.

[0135] (3) If there are 1 or 2 positive droplets, it is indicated that the result is in the gray area, and retesting is recommended. If the retesting still has <3 positive droplets, it is determined that the result is negative, and it is reported that nothing is detected.

[0136] The kit of the present application

[0137] The present application provides a kit for detecting Candida glabrata, Candida, Candida tropicalis, and Cryptococcus. The kit comprises PCR primer probes (including internal reference primer probes), a reaction system, reaction conditions, a negative control DNA sample, and a positive control DNA sample.

[0138] Primers and probes: The oligonucleotide fragments and modified oligonucleotide fragments shown in Table 1 are synthesized and dissolved in TE (10μM).

[0139] PCR reagents: DNA polymerase uses 5×HS Taq Buffer with Mg 2+ Reaction system.

[0140] Control: The blank control is water, the negative control is normal human leukocyte genome (tgDNA) with a copy concentration of 1000 copies / μL. The positive control is a mixture of Candida glabrata, Candida, C. tropicalis and Cryptococcus genome. The copy number of all target sites of the positive control solution is 50 copies / μL.

[0141] The reaction conditions are as described above.

[0142] The kit of the present application can be used to quickly and accurately detect the following blood stream infection pathogens: Candida glabrata, Candida, C. tropicalis and Cryptococcus.

[0143] The main advantages of the present application include:

[0144] (a) Compared with the prior art blood culture, MALD-TOF MS, NGS, etc., the present application uses Taqman probe and combines digital PCR method, which can solve the problems of low sensitivity, poor specificity, high requirements for sample type and quality, and complex positive interpretation method.

[0145] (b) High sensitivity: Since the present method uses a digital PCR platform, the reaction system can be divided into about 20000 small reactions, which can theoretically detect a single copy of the mutation, and has a sensitivity advantage that other technologies cannot match. The detection method of the present application can achieve a minimum detection limit of 5 copies / reaction through verification.

[0146] (c) Strong specificity: The designed specific primers and probes are specific to the specific sequences of Candida glabrata, Candida, C. tropicalis and Cryptococcus, and can specifically amplify the target position.

[0147] (d) The requirements for sample type and quality are relaxed, and the anti-interference ability is strong. Due to the high sensitivity of the present application, the applicable sample type is peripheral blood sample (this sample is easier to obtain, but the DNA content is low and broken); and due to the uniqueness of its digital PCR platform, the reaction system can be divided into about 20000 small systems, and at the same time, the interfering substances can also be divided into about 20000 parts, which can greatly reduce the influence of interfering substances on the reaction, and of course, more complex background samples can be detected. This is something that other platforms cannot do.

[0148] (e) Simple positive interpretation method: Since the present application uses an absolute quantitative method, there is no need to set a standard curve, and the result can be determined according to the two-dimensional fluorescence chart whether it contains the target mutation template. The interpretation is as follows:

[0149] (1) If the positive droplet is ≥3, and the clustering and positive control are consistent, it indicates a positive result, and the infection pathogen and copy number are directly reported;

[0150] (2) If there is no positive droplet, and the clustering and negative control are consistent, it is suggested that the result is negative, and the report is not detected;

[0151] (3) If there is one or two positive droplets, it is suggested that the result is in the gray area, and retesting is recommended; if the retesting still has less than three positive droplets, it is determined that the result is negative, and the report is not detected.

[0152] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application. The experimental methods in the following examples are not specified, and the methods are generally performed according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0153] Example 1 Digital PCR method for detecting Candida glabrata, Candida, Candida tropicalis, Cryptococcus

[0154] 1.1 Primer and probe design

[0155] Primer and probe design for detecting pathogens: A plurality of groups of primers and probes were designed according to the specific genes of Candida glabrata, Candida, Candida tropicalis, and Cryptococcus (Table A), and the sequences with no mutual influence and optimal amplification efficiency were finally selected as shown in Table 2. Among them, Candida glabrata and Candida tropicalis belong to the genus Candida, but these two strains have a high detection rate in clinical practice, and the treatment method is different from that of general Candida, therefore, primers and probes capable of specifically detecting these two strains are designed and listed.

[0156] Primer and probe design for detecting pathogens: A plurality of groups of primers and probes were designed according to the specific genes of Candida glabrata, Candida, Candida tropicalis, and Cryptococcus (Table A), and the sequences with no mutual influence and optimal amplification efficiency were finally selected as shown in Table 2. Among them, Candida glabrata and Candida tropicalis belong to the genus Candida, but these two strains have a high detection rate in clinical practice, and the treatment method is different from that of general Candida, therefore, primers and probes capable of specifically detecting these two strains are designed and listed.

[0157] Table 2

[0158]

[0159] Note: S represents G / C degenerate base.

[0160] Table A Specific genes of Candida glabrata, Candida, Candida tropicalis, and Cryptococcus and sequence information thereof

[0161]

[0162]

[0163] 1.2 PCR system:

[0164] 5x 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 (10μM) 1μL, SEQ ID NO: 6 (10μM) 0.5μL, SEQ ID NO: 7 (10μM) 1μL, SEQ ID NO: 8 (10μM) 1μL, SEQ ID NO: 9 (10μM) 0.5μL, SEQ ID NO: 10 (10μM) 1μL, SEQ ID NO: 11 (10μM) 1μL, SEQ ID NO: 12 (10μM) 0.5μL, template 5μL, and water to 30μL.

[0165] 1.3 PCR program: 95℃ 10min, 40 cycles (95℃ 30s, 56℃ 15s, 72℃ 15s).

[0166] 1.4 Chip scanning

[0167] After testing, Candida glabrata, Candida (Candida albicans), Candida tropicalis, Cryptococcus (Cryptococcus neoformans) and internal reference can be effectively detected, as shown in Figures 1-5 In addition, 9 kinds of Candida commonly seen in clinical samples were detected, and the detection results are shown in Table 3:

[0168] Table 3

[0169]

[0170] By comparing the single and multiplex results of each target point, it can be seen that there is no difference between the amplification concentration results of single and multiplex (Table 4), therefore, there is no mutual influence between primer probes that leads to reduced PCR amplification efficiency, see Figures 6-13 .

[0171] Table 4

[0172]

[0173] Example 2 Sensitivity verification of digital PCR method for detecting Candida glabrata, Candida, Candida tropicalis, Cryptococcus

[0174] 2.1 Experimental method

[0175] The strains detected include Candida and Cryptococcus. The Candida includes Candida glabrata, Candida tropicalis, Candida paratropicalis, Candida dubliniensis, Candida guilliermondii, Candida parapsilosis, Candida kefyr, and Candida auris. The Cryptococcus includes Cryptococcus neoformans and Cryptococcus gattii.

[0176] According to Example 1, the copy number of each pathogen template (copies / μL) was calculated, and each pathogen template was diluted to 8 copies / μL, 4 copies / μL, 2 copies / μL, and 1 copies / μL with TE to prepare a gradient dilution template.

[0177] Verification of the digital PCR system: 5x HS Taq Buffer with Mg 2+ 6 μL, dNTPs (10 mM each) 0.75 μL, HotStart 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 water to 30 μL.

[0178] In the sample preparation area, the templates were added in the following order: blank control, negative control, and gradient dilution template. The blank control was water, the negative control was tgDNA, and the gradient dilution template was 8 copies / μL, 4 copies / μL, 2 copies / μL, and 1 copies / μL of each pathogen genome.

[0179] The PCR reaction system was generated into droplets in the same way as Example 1. PCR was performed according to the PCR program: 95℃ 10min, 40 cycles (95℃ 30s, 56℃ 15s, 72℃ 15s). Scanning was started according to the instrument requirements.

[0180] 2.1 Experimental results

[0181] The digital PCR results are shown in Figures 14-23 The digital PCR method of the present application uses water or tgDNA as a template, and has a clean background without pollution. In addition, the digital PCR method of the present application can effectively detect positive points when detecting 1 copies / μL of template, i.e., the minimum detection sensitivity of the digital PCR method of the present application is 5 copies / reaction.

[0182] Example 3 Verification of the specificity of the digital PCR method for detecting Candida glabrata, Candida, Candida tropicalis, and Cryptococcus

[0183] 3.1 Experimental method

[0184] Primers and probes for different pathogens were designed, and the template copy number (copies / μL) of each pathogen was calculated according to Example 1. Each pathogen template was diluted to 10000 copies / μL with TE. Then, each pathogen was mixed in equal proportions according to 5 groups (a total of 8 groups or 8 samples), as shown in Table 5.

[0185] Table 5

[0186]

[0187] Verification of the digital PCR system: 5×HS Taq Buffer with Mg 2+6 μL, dNTPs (10 mM each) 0.75 μL, HotStart 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 30 μL.

[0188] The templates were added in the following order in the sample preparation area: blank control, negative control, mixed pathogen template. The blank control was water, the negative control was tgDNA, and the mixed pathogen template was as shown in the above table.

[0189] The PCR reaction system was generated into droplets in the same way 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). The plate was read according to the instrument requirements.

[0190] 3.2 Experimental results

[0191] The digital PCR results are shown in the accompanying Figure 24 The digital PCR method of the present application uses water or tgDNA as a template, and has a clean background without contamination. In addition, the digital PCR method of the present application has no cross-reaction when detecting clinical common pathogens other than the target to be detected, at a concentration of 2000 copies / μL, showing good specificity.

[0192] All the documents mentioned in the present application are incorporated by reference in the present application, as if each document is individually incorporated by reference. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the above teachings of the present application, and these equivalent forms also fall within the scope of the appended claims of the present application.

Claims

1. A primer probe combination for detecting a blood stream infection pathogen, characterized by, The primer probe combination comprises: (a) upper and lower primers and probes for detecting Candida as shown in SEQ ID NO: 4-6; (b) upper and lower primers and probes for detecting C. glabrata as shown in SEQ ID NO: 1-3; (c) upper and lower primers and probes for detecting C. tropicalis as shown in SEQ ID NO: 7-9; and (d) upper and lower primers and probes for detecting Cryptococcus as shown in SEQ ID NO: 10-12; wherein the fluorescent reporter group of the Candida probe is HEX; the fluorescent reporter group of the C. glabrata probe is FAM; the fluorescent reporter group of the C. tropicalis probe is ROX; and the fluorescent reporter group of the Cryptococcus probe is CY5.

2. The primer probe combination of claim 1, wherein, The Candida comprises C. albicans, C. glabrata, C. tropicalis, C. pseudotropicalis, C. dubliniensis, C. guilliermondii, C. parapsilosis, C. kefyr, and C. auris.

3. The primer probe combination of claim 1, wherein The primer probe combination further comprises (e) upper and lower primers and probes for detecting an internal reference gene as shown in SEQ ID NO: 17-19.

4. The primer probe combination of claim 1, wherein, The Cryptococcus comprises C. neoformans and C. gattii.

5. Use of a primer probe combination according to claim 1, characterized in that The kit for preparing a reagent for detecting a pathogen of bloodstream infection, wherein the pathogen comprises Candida and Cryptococcus; wherein the Candida comprises C. albicans, C. glabrata, C. tropicalis, C. pseudotropicalis, C. dubliniensis, C. guilliermondii, C. parapsilosis, C. kefyr, and C. auris; and the Cryptococcus comprises C. neoformans and C. gattii.

6. A kit for detecting a blood stream infection pathogen, characterized by, The kit comprises the primer probe combination of claim 1.

7. The kit of claim 6, wherein The kit further comprises a negative quality control and a positive quality control.

8. The kit of claim 7, wherein The negative quality control is normal human leukocyte genomic DNA.

9. Use of a primer probe combination according to claim 1 or a kit according to claim 6, characterized in that, The reagent for preparing a reagent for detecting a pathogen of bloodstream infection, wherein the pathogen comprises Candida and Cryptococcus; wherein the Candida comprises C. albicans, C. glabrata, C. tropicalis, C. pseudotropicalis, C. dubliniensis, C. guilliermondii, C. parapsilosis, C. kefyr, and C. auris; and the Cryptococcus comprises C. neoformans and C. gattii.

10. Use according to claim 9, characterized in that, The detection comprises the following steps: (i) extracting free DNA in a 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) reading the fluorescence data and calculating the copy concentration of the target sequence to obtain a detection result.

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