Method and kit for detecting bloodstream infection pathogens and application

Abstract

The invention provides a method and a kit for detecting blood flow infection pathogens and application of the method and the kit. Specifically, the invention provides a primer probe combination for detecting bloodstream infection pathogens, and the primer probe combination comprises: (a) upstream and downstream primers and probes for detecting candida as shown in SEQ ID NO: 4-6; (b) upstream and downstream primers and probes for detecting candida glabrata as shown in SEQ ID NO: 1-3; (c) upstream and downstream primers and probes for detecting candida tropicalis as shown in SEQ ID NO: 7-9; 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 cryptococcus. The primer probe combination provided by the invention can be used for realizing rapid, high-sensitivity and high-specificity detection of blood flow infection pathogens.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnostics, and specifically, to a method, a kit and an application for detecting pathogens causing bloodstream infection. Background Art

[0002] Bloodstream infection (BSI) refers to the presence of pathogenic microorganisms in a patient's blood. The pathogenic microorganisms causing bloodstream infection include bacteria, fungi, viruses, 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 procedures, 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 causing bloodstream infection is identified and the correct antibacterial drug treatment is selected, the mortality of the patient will decrease by about 7%. Therefore, it is crucial to quickly and timely identify the pathogenic bacteria causing bloodstream infection.

[0004] For a long time, there have been many problems in the clinical diagnosis and treatment of BSI, such as the need for continuous improvement in early rapid diagnosis, precise treatment, etc. 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) technology, the detection cost is high, so it is not suitable for the rapid detection required by general bloodstream infection patients, and NGS needs to first enrich the DNA of the detected sample by PCR method, so there will be amplification preference for some segments, resulting in missed detection and insufficient accuracy problems.

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

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

[0007] In the first aspect of the present invention, a primer-probe combination (or primer-probe composition) for detecting pathogens causing bloodstream infection is provided, and the primer-probe combination (or primer-probe composition) includes: (a) Upstream and downstream primers and probes for detecting Candida spp. as shown in SEQ ID NO: 4 - 6; (b) Upstream and downstream primers and probes for detecting Candida glabrata as shown in SEQ ID NO: 1 - 3; (c) Upstream and downstream primers and probes for detecting Candida tropicalis as shown in SEQ ID NO: 7 - 9; and (d) Upstream and downstream primers and probes for detecting Cryptococcus spp. as shown in SEQ ID NO: 10 - 12.

[0008] In another preferred example, the primer - probe combination further includes primers and probes for detecting other bloodstream - infecting pathogens.

[0009] In another preferred example, the primer - probe combination and the primer - probe composition have the same meaning and can be used interchangeably.

[0010] In another preferred example, the primer - probe combination further includes: (e) Upstream and downstream primers and probes for detecting an internal reference (or internal reference gene) as shown in SEQ ID NO: 17 - 19.

[0011] In another preferred example, the primers and probes for detecting the internal reference are designed based on specific conserved regions of the human EGFR gene.

[0012] In another preferred example, the Candida spp. includes: Candida albicans, Candida glabrata, Candida tropicalis, Candida pseudotropicalis, Candida dubliniensis, Candida guilliermondii, Candida parapsilosis, Candida krusei, and Candida auris.

[0013] In another preferred example, the Cryptococcus spp. includes Cryptococcus neoformans and Cryptococcus gattii.

[0014] 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.

[0015] In this article, "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 they do not affect the detection of each other, that is, they can be detected using different channels. For example, FAM, HEX, ROX, CY5, and Q705 can be used. The absorbance values of these groups are not close, and different channels can be selected, so they do not interfere with each other.

[0016] In another preferred example, the fluorescent reporter group of the Candida spp. probe is HEX; the fluorescent reporter group of the Candida glabrata probe is FAM; the fluorescent reporter group of the Candida tropicalis probe is ROX; the fluorescent reporter group of the Cryptococcus spp. probe is CY5.

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

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

[0019] In another preferred embodiment, the fluorescent quenching group is selected from the group consisting of: BHQ1, BHQ2, BHQ3.

[0020] In another preferred embodiment, the quenching group at the 3'-end of the Candida glabrata and the Candida genus probe is BHQ1; the quenching group at the 3'-end of the Candida tropicalis probe and the Cryptococcus genus probe is BHQ2.

[0021] In another preferred embodiment, the quenching group at the 3'-end of the internal reference probe is BHQ3.

[0022] In another preferred embodiment, each component of the primer-probe combination is present in a separate package.

[0023] In another preferred embodiment, each component of the primer-probe combination is present in the same package.

[0024] In another preferred embodiment, each component of the combination exists in a mixed form.

[0025] 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.

[0026] In the second aspect of the present invention, there is provided the use of the primer-probe combination as described in the first aspect of the present invention for preparing a kit for detecting blood-stream infection pathogens; wherein, the pathogens include: Candida genus, Candida glabrata, Candida tropicalis and Cryptococcus genus.

[0027] In another preferred embodiment, the Candida genus includes: Candida albicans, Candida glabrata, Candida tropicalis, Candida pseudotropicalis, Candida dubliniensis, Candida guilliermondii, Candida parapsilosis, Candida krusei and Candida auris.

[0028] In another preferred embodiment, the Cryptococcus genus includes Cryptococcus neoformans and Cryptococcus gattii.

[0029] In the third aspect of the present invention, there is provided a kit for detecting blood-stream infection pathogens, the kit comprising the primer-probe combination as described in the first aspect of the present invention.

[0030] In another preferred embodiment, the kit further includes a negative control product and a positive control product.

[0031] In another preferred example, the negative control product is at least one of DEPC-H2O, normal saline, internal standard gene pseudovirus, and normal human leukocyte genomic DNA.

[0032] In another preferred example, the negative control product is normal human leukocyte genomic DNA (tgDNA).

[0033] In another preferred example, the copy number concentration of the tgDNA is 1000 copies / μL.

[0034] In another preferred example, the positive control product is at least one of fragment plasmids or fragment DNAs of Candida, Candida glabrata, Candida tropicalis, and Cryptococcus.

[0035] In another preferred example, the positive control product is a mixture of the genomes of Candida, Candida glabrata, Candida tropicalis, and Cryptococcus.

[0036] In another preferred example, the copy number of all target sites in the positive control product is 50 copies / μL.

[0037] 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 NO: 17-19.

[0038] In another preferred example, the kit further includes a PCR reaction solution.

[0039] In another preferred example, the PCR reaction solution includes at least one of dNTP, PCR buffer, Mg 2+ and DNA polymerase.

[0040] In another preferred example, the DNA polymerase is 5×HS Taq Buffer with Mg 2+ reaction system.

[0041] In another preferred example, the kit further contains an instruction manual, and the instruction manual indicates that the kit is used for detecting blood stream infection pathogens.

[0042] In another preferred example, the kit further includes a blank control, and the blank control is ultrapure water.

[0043] 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.

[0044] In another preferred example, the detection includes the following steps: (i) Extract free DNA from the sample to be tested; (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 test result.

[0045] In another preferred embodiment, in step (iii), the determination of the test result is as follows: (1) If the number of positive droplets ≥ 3 and the clustering is consistent with the positive control, it indicates a positive result, and the infecting pathogen and the copy number are directly reported; (2) If there are no positive droplets and the clustering is consistent with the negative control, it indicates a negative result, and it is reported that no detection is found; (3) If there is 1 or 2 positive droplets, it indicates a gray zone, and re-examination is recommended; if there are still < 3 positive droplets after re-examination, it is determined as a negative result, and it is reported that no detection is found.

[0046] In another preferred embodiment, the sample to be tested is selected from the group consisting of whole blood, plasma, serum, or a combination thereof.

[0047] In the fifth aspect of the present invention, a method for detecting pathogens in bloodstream infections is provided, comprising the following steps: (i) Extract free DNA from the sample to be tested; (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 test result.

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

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

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

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

[0052] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (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

[0053] Figure 1 The figure shows the detection result graph of Candida glabrata positive detected by using the multiplex primer-probe combination of the present invention.

[0054] Figure 2 The figure shows the detection result graph of Candida genus (Candida albicans) positive detected by using the multiplex primer-probe combination of the present invention.

[0055] Figure 3 The figure shows the detection result graph of Candida tropicalis positive detected by using the multiplex primer-probe combination of the present invention.

[0056] Figure 4 The figure shows the detection result graph of Cryptococcus genus (Cryptococcus neoformans) positive detected by using the multiplex primer-probe combination of the present invention.

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

[0058] Figure 6 The figure shows the concentration result of amplifying the target gene of Candida glabrata by using the multiplex primer-probe combination.

[0059] Figure 7 The figure shows the concentration result of single amplification of the target gene of Candida glabrata.

[0060] Figure 8 The figure shows the concentration result of amplifying the target gene of Candida genus (Candida albicans) by using the multiplex primer-probe combination.

[0061] Figure 9 The figure shows the concentration result of single amplification of the target gene of Candida genus (Candida albicans).

[0062] Figure 10 The figure shows the concentration result of amplifying the target gene of Candida tropicalis by using the multiplex primer-probe combination.

[0063] Figure 11 The figure shows the concentration result of single amplification of the target gene of Candida tropicalis.

[0064] Figure 12Shows the concentration results of amplifying the target gene of Cryptococcus (Cryptococcus neoformans) using a multiplex primer-probe combination.

[0065] Figure 13 Shows the concentration results of single amplification of the target gene of Cryptococcus (Cryptococcus neoformans).

[0066] Figure 14 Shows the detection sensitivity of Candida glabrata in Example 2.

[0067] Figure 15 Shows the detection sensitivity of Candida (Candida pseudotropicalis) in Example 2.

[0068] Figure 16 Shows the detection sensitivity of Candida (Candida dubliniensis) in Example 2.

[0069] Figure 17 Shows the detection sensitivity of Candida (Candida guilliermondii) in Example 2.

[0070] Figure 18 Shows the detection sensitivity of Candida (Candida parapsilosis) in Example 2.

[0071] Figure 19 Shows the detection sensitivity of Candida (Candida krusei) in Example 2.

[0072] Figure 20 Shows the detection sensitivity of Candida (Candida auris) in Example 2.

[0073] Figure 21 Shows the detection sensitivity of Candida tropicalis in Example 2.

[0074] Figure 22 Shows the detection sensitivity of Cryptococcus (Cryptococcus neoformans) in Example 2.

[0075] Figure 23 Shows the detection sensitivity of Cryptococcus (Cryptococcus gattii) in Example 2.

[0076] Figure 24 Shows the specific detection results of mixed pathogens in Example 3.

[0077] General description of the drawings: The gray markings present in the drawings represent supplementary explanations of the figures, used to prompt the specific single-channel fluorescence information of the individual figures below, that is, the signal intensity measured by the droplets in a certain specific fluorescence detection channel (such as FAM, HEX, ROX, CY5). Detailed implementation mode

[0078] After extensive and in-depth research and extensive screening, 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: Candida spp., Candida glabrata, Candida tropicalis, and Cryptococcus spp. Experiments of the present invention showed that there was no difference in the amplification concentration results between single and multiplex PCR, so there was no mutual influence among the primers and probes of the present invention that would lead to a decrease in PCR amplification efficiency. Moreover, the minimum detection sensitivity of the digital PCR method of the present invention was 5 copies / reaction, and the specificity was good. On this basis, the present invention was completed.

[0079] Term 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.

[0080] As used herein, the term "comprising" or "including" can be open-ended, semi-closed, and closed-ended. In other words, the term also includes "consisting essentially of...", or "consisting of...".

[0081] As used herein, unless otherwise indicated, 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 (e.g., one-tenth and one-hundredth of an integer).

[0082] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0083] Bloodstream infection Bloodstream infection (BSI) refers to an infection caused by pathogenic microorganisms entering the bloodstream, 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.

[0084] There is a 40% probability that BSI will develop into sepsis. There are 6.12 million new cases of sepsis in China every year, the 90-day mortality rate is 33.5%, the average length of stay in the ICU is 32 days, and the average treatment cost is 300,000 yuan. Research has found that for septic shock patients, the survival rate decreases by 7.6% for every 1-hour delay in treatment after hypotension occurs, and the survival rate at a median delay of 6 hours is 42%.

[0085] 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 Luminex. However, these molecular detection methods are all based on positive blood culture isolates for detection. There are also some mNGS-based detection methods in China, but the detection time of mNGS 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.

[0086] The pathogen laboratory detection of BSI takes a long time and is difficult to diagnose, which is a major challenge that needs to be urgently 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.

[0087] The multiplex digital PCR (dPCR) rapid detection method randomly divides the fluorescence quantitative reaction system into tens of thousands of independent tiny reaction units, and realizes the absolute quantification of nucleic acid molecules according to the Poisson distribution and the proportion of positive droplets. As the third-generation PCR technology, dPCR has higher sensitivity than fluorescence quantitative PCR, strong anti-interference ability, can achieve absolute quantification, and is suitable for the highly difficult detection of BSI with low levels of pathogenic microorganisms, complex blood components, and high human background DNA signals. The innovative rapid blood stream infection pathogen detection technology based on multiplex digital PCR has a detection limit as low as 5-10 copies / reaction, and the reporting cycle is shortened from the current 2-3 days of blood culture to 4 hours, which will effectively solve the worldwide problems such as low detection rate and long reporting cycle of current blood stream infection pathogens.

[0088] Candida spp., Candida glabrata, Candida tropicalis and Cryptococcus spp. Candida spp. is a type of yeast-like fungus belonging to the Ascomycota in the fungal kingdom. Candida spp. includes a variety of pathogenic and non-pathogenic species, and the most common one is Candida albicans. Candida glabrata and Candida tropicalis both belong to Candida spp. Candida spp. is one of the main pathogens of hospital-acquired bloodstream infections.

[0089] Candida glabrata is one of the common pathogens of hospital-acquired bloodstream infections (candidemia), commonly found in immunocompromised patients (such as cancer patients, organ transplant recipients) or patients who have long-term use of broad-spectrum antibiotics or indwelling central venous catheters.

[0090] Candida tropicalis is one of the common pathogens of candidemia, with relatively strong virulence, and is common in patients with hematological malignancies. After infection, it may lead to severe systemic infections such as sepsis and multiple organ failure.

[0091] Cryptococcus is a type of yeast-like fungus belonging to the Basidiomycota phylum. The most common pathogenic species in the genus Cryptococcus are Cryptococcus neoformans and Cryptococcus gattii. Cryptococcus can disseminate through the bloodstream, causing cryptococcosis, and further leading to central nervous system infections (such as cryptococcal meningitis). The mortality rate of cryptococcosis is relatively high in immunocompromised patients.

[0092] The detection method of the present invention The present invention provides a method for detecting pathogens causing bloodstream infections, the method comprising the following steps: 1) Design specific primer-probes (including primer and probe for internal reference) according to the gene sequences of the pathogens. The pathogens include Candida glabrata, Candida spp., Candida tropicalis, and Cryptococcus. Perform digital PCR detection using the above primers and probes, and optimize the best detection conditions for the PCR system. Apply this method to detect patient blood samples. The specific primer-probe sequences are shown in Table 1 below.

[0093] Table 1

[0094] Note: S represents G / C degenerate base.

[0095] Among them, the 5' end of the probe sequence is also connected to a fluorescent reporter gene, and the 3' end is also connected to a fluorescent quenching group. The fluorescent reporter group is selected from the group consisting of: FAM, HEX, ROX, CY5, and Q705; the fluorescent quenching group is selected from the group consisting of: BHQ1, BHQ2, and BHQ3.

[0096] 2) Extract nucleic acids from the sample. Select a method suitable for the sample type to extract nucleic acids, and 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 blood cell stabilization technology should be selected.

[0097] 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.

[0098] Third, extraction. Use a special extraction kit for plasma cell-free nucleic acids and extract according to the instructions.

[0099] 3) Digital PCR.

[0100] First, configuration of the PCR system. Configure the PCR system in the reagent preparation area according to the following table: 5×HS Taq Buffer with Mg 2+ 6 μL, dNTPs (10 mM each) 0.75 μL, HotStart 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.

[0101] Preferably, the final concentration of each primer is 0.33 μM, and the final concentration of each probe is 0.17 μM.

[0102] Second, add the template. Add the template in the sample preparation area in the following order: 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 copy number of all target sites in the positive control solution is 50 copies / μL. The blank control is ultrapure water.

[0103] Third, generation of droplets. Generate droplets according to the instrument requirements.

[0104] Fourth, PCR. After the annealing temperature optimization experiment, it was found that the digital PCR effect was optimal at 56°C annealing for 15 s. Perform PCR according to the following program: 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s), with a temperature rise and fall rate of 2°C / s.

[0105] Fifth, scanning. Judge whether the sample is positive or negative according to the scanning result.

[0106] The determination of the test results is as follows: (1) If there are ≥3 positive droplets and the clustering is consistent with the positive control, it indicates a positive result, and the infectious pathogen and the copy number are directly reported; (2) If there are no positive droplets and the clustering is consistent with the negative control, it indicates a negative result, and it is reported that no detection is found; (3) If there is 1 or 2 positive droplets, it indicates a gray zone, and retesting is recommended; if there are still <3 positive droplets after retesting, it is determined as a negative result, and it is reported that no detection is found.

[0107] The kit of the present invention The present invention provides a kit for detecting Candida glabrata, Candida spp., Candida tropicalis and Cryptococcus spp. The kit includes PCR primer probes (including internal reference primer probes), reaction systems, reaction conditions, negative control DNA samples, and positive control DNA samples.

[0108] Primers and probes: Synthesize the oligonucleotide fragments and modified oligonucleotide fragments shown in Table 1 and dissolve them in TE (10 μM).

[0109] PCR reagent: Use 5×HS Taq Buffer with Mg for DNA polymerase 2+ Reaction system.

[0110] 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 Candida glabrata, Candida spp., Candida tropicalis, and Cryptococcus spp. The copy number of all target sites in the positive control solution is 50 copies / μL.

[0111] The reaction conditions are as described above.

[0112] The kit of the present invention can be used to quickly and accurately detect the following bloodstream infection pathogens: Candida glabrata, Candida spp., Candida tropicalis, and Cryptococcus spp.

[0113] The main advantages of the present invention include: (a) Compared with the existing technologies such as blood culture, MALD-TOF MS, and NGS, the present invention adopts Taqman probes and combines digital PCR method, which can solve the problems of low sensitivity, poor specificity, high requirements for the type and quality of samples, and complex positive interpretation methods.

[0114] (b) High sensitivity: Since this method adopts a digital PCR platform, it can divide the reaction system into about 20,000 tiny reactions, and theoretically can detect single-copy mutations, having a sensitive 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.

[0115] (c) High specificity: The designed specific primer-probes are respectively targeted at the specific sequences of Candida glabrata, Candida spp., Candida tropicalis, and Cryptococcus spp., and can specifically amplify the target positions.

[0116] (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 samples (such samples are relatively easy to obtain, but have low DNA content and are fragmented); moreover, due to the uniqueness of its digital PCR platform, that is, 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, and of course can detect samples with more complex backgrounds. This cannot be achieved by other platforms.

[0117] (e) Simple positive interpretation method: Since the present invention adopts an absolute quantification method, there is no need to set a control standard curve, and the result can be determined whether it contains the target mutant template according to the two-dimensional fluorescence map. The interpretation 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.

[0118] 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 usually carried out under conventional conditions, such as 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 stated, percentages and parts are weight percentages and weight parts.

[0119] Example 1 Detection of Candida glabrata, Candida spp., Candida tropicalis, and Cryptococcus spp. by Digital PCR 1.1 Primer and Probe Design Primer and probe design for pathogen detection: Multiple sets of primers and probes were designed based on the specific genes of Candida glabrata, Candida spp., Candida tropicalis, and Cryptococcus spp. (Table A), and finally, sequences that do not interfere with each other and have excellent amplification efficiency were selected as shown in Table 2 below. Among them, both Candida glabrata and Candida tropicalis belong to the genus Candida, but these two strains have a high detection rate clinically, and the treatment methods are different from those of general Candida. Therefore, the present invention designed and listed primers and probes that can specifically detect these two strains.

[0120] Primer and probe design for internal reference: Primers and probes were screened from multiple human genes, aiming to have no interference with the pathogen primers and probes for detection above and have excellent amplification efficiency. Finally, the internal reference sequences in Table 2 below were obtained as the internal reference sequences of the kit. The upstream and downstream primers and probes for detecting the internal reference were designed based on specific conserved regions of the human EGFR gene.

[0121] Table 2

[0122] Note: S represents a G / C degenerate base.

[0123] Table A Specific genes and their sequence information of Candida glabrata, Candida spp., Candida tropicalis, and Cryptococcus spp.

[0124]

[0125] 1.2 PCR system: 5×HS Taq Buffer with Mg 2+ 6 μL, dNTPs (10 mM each) 0.75 μL, HotStart TaqDNA Polymerase 0.2 μL, SEQ ID NO: 1 (10 μM) 1 μL, SEQ ID NO: 2 (10 μM) 1 μL, SEQ ID NO:3 (10 μM) 0.5 μL, SEQ ID NO: 4 (10 μM) 1 μL, SEQ ID NO: 5 (10 μM) 1 μL, SEQ ID NO: 6 (10 μM) 0.5 μL, SEQ ID NO: 7 (10 μM) 1 μL, SEQ ID NO: 8 (10 μM) 1 μL, SEQ ID NO: 9 (10 μM) 0.5 μL, SEQ ID NO: 10 (10 μM) 1 μL, SEQ ID NO: 11 (10 μM) 1 μL, SEQ ID NO: 12 (10 μM) 0.5 μL, template 5 μL, made up to 30 μL with water.

[0126] 1.3 PCR procedure: 10 min at 95°C, 40 cycles (30 s at 95°C, 15 s at 56°C, 15 s at 72°C).

[0127] 1.4 Chip scanning After testing, it can effectively detect Candida glabrata, Candida spp. (Candida albicans), Candida tropicalis, Cryptococcus spp. (Cryptococcus neoformans), and internal reference, as Figures 1 - 5 shown. In addition, when detecting 9 species of Candida spp. in common clinical samples, the detection results are shown in Table 3 below: Table 3

[0128] 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 causes a decrease in PCR amplification efficiency. For details, see Figures 6 - 13 .

[0129] Table 4

[0130] Example 2 Verification of the sensitivity of digital PCR for detecting Candida glabrata, Candida spp., Candida tropicalis, and Cryptococcus spp. 2.1 Experimental method The strains to be detected include Candida spp. and Cryptococcus spp. The Candida spp. include: Candida glabrata, Candida tropicalis, Candida pseudotropicalis, Candida dubliniensis, Candida guilliermondii, Candida parapsilosis, Candida krusei, and Candida auris. The Cryptococcus spp. include Cryptococcus neoformans and Cryptococcus gattii.

[0131] Calculate the template copy number (copies / μL) of each pathogen according to Example 1, and dilute each pathogen template to 8 copies / μL, 4 copies / μL, 2 copies / μL, and 1 copies / μL with TE to make gradient-diluted templates.

[0132] Verify 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, make up to 30 μL with water.

[0133] In the sample preparation area, add the templates in the following order: blank control, negative control, gradient-diluted templates. The blank control is water, the negative control is tgDNA, and the gradient-diluted templates are genomic DNAs of each pathogen at 8 copies / μL, 4 copies / μL, 2 copies / μL, and 1 copy / μL.

[0134] 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 scanning according to the instrument requirements.

[0135] 2.1 Experimental results The digital PCR results are shown in Figures 14 - 23 , the digital PCR method of the present invention uses water or tgDNA as the template, with a clean background and no contamination. In addition, the digital PCR method of the present invention can effectively detect positive points when detecting a 1 copy / μL template, that is, the lowest detection sensitivity of the digital PCR method of the present invention is 5 copies / reaction.

[0136] Specific verification of the digital PCR method of Example 3 for detecting Candida glabrata, Candida spp., Candida tropicalis, and Cryptococcus spp. 3.1 Experimental method Design primers and probes for different pathogens, calculate the copy number (copies / μL) of each pathogen template according to Example 1, and dilute each pathogen template to 10000 copies / μL with TE. Then mix each pathogen in equal proportions according to 5 in a group (a total of 8 groups or 8 samples), and the combinations are shown in Table 5 below.

[0137] Table 5

[0138] Verification of 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, make up to 30 μL with water.

[0139] Add templates in the following order in the sample preparation area: blank control, negative control, mixed pathogen template. The blank control is water, the negative control is tgDNA, and the mixed pathogen template is as shown in the above table.

[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] 3.2 Experimental results The results of digital PCR are shown in the appendix Figure 24 , for the digital PCR method of the present invention, using water or tgDNA as the template, the background is clean and there is no pollution. In addition, when detecting common clinical pathogens other than the detection target by the digital PCR method of the present invention, there is no cross-reaction at a concentration of 2000 copies / μL, indicating good specificity.

[0142] All documents mentioned in the present invention are cited herein by reference as if each document was individually cited 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 the present application.

Claims

1. A primer-probe combination for detecting blood-stream infection pathogens, characterized in that, The primer-probe combination includes: (a) Upstream and downstream primers and a probe for detecting Candida genus as shown in SEQ ID NO: 4-6; (b) Upstream and downstream primers and a probe for detecting Candida glabrata as shown in SEQ ID NO: 1-3; (c) Upstream and downstream primers and a probe for detecting Candida tropicalis as shown in SEQ ID NO: 7-9; and (d) Upstream and downstream primers and a probe for detecting Cryptococcus genus as shown in SEQ ID NO: 10-12.

2. The primer-probe combination according to claim 1, wherein The Candida genus includes: Candida albicans, Candida glabrata, Candida tropicalis, Candida pseudotropicalis, Candida dubliniensis, Candida guilliermondii, Candida parapsilosis, Candida krusei, and Candida auris.

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 a reference gene as shown in SEQ ID NO: 17-19.

4. The primer-probe combination according to claim 1, wherein The fluorescence reporter group of the Candida genus probe is HEX; the fluorescence reporter group of the Candida glabrata probe is FAM; the fluorescence reporter group of the Candida tropicalis probe is ROX; the fluorescence reporter group of the Cryptococcus genus 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: Candida genus, Candida glabrata, Candida tropicalis, and Cryptococcus genus.

6. A kit for detecting pathogens causing bloodstream infections, 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 6, wherein The positive control product is a mixture of genomes of Candida genus, Candida glabrata, Candida tropicalis, and Cryptococcus genus; the negative control product is genomic DNA (tgDNA) of normal human leukocytes.

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

10. The use according to claim 9, characterized in that, The detection includes the following steps: (i) Extracting free DNA in 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 or the kit as described in claim 6; (iii) Reading the fluorescence data and calculating the copy concentration of the target sequence to obtain the detection result.

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