Primer probe and kit for rapid detection of common candida of blood flow infection and application of primer probe and kit

Abstract

The invention discloses a primer probe and a kit for rapidly detecting common candida of blood flow infection and application, and particularly relates to a candida universal primer and a Taqman-MGB probe which are required for implementing a fluorescent quantitative PCR (Polymerase Chain Reaction) technology, as well as a red blood cell lysis buffer, a white blood cell lysis buffer and the like. The lysate can efficiently lyse red blood cells and white blood cells in peripheral blood, and the primers and the probes can specifically amplify DNA of candida albicans, candida glabrata, candida parapsilosis, candida tropicalis and candida krusei. The kit takes whole blood as a detection sample and has the characteristics of high sensitivity, good precision, high detection speed and the like, so that early diagnosis and timely treatment of diseases are assisted.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a primer probe and a kit and application for rapid detection of common Candida fungi in bloodstream infection, and more particularly to universal primers and Taqman-MGB probes for Candida fungi required for the implementation of fluorescent quantitative PCR technology, as well as red blood cell lysate and white blood cell lysate. Background Art

[0002] Bloodstream infection (BSI) refers to a systemic infectious disease in which various pathogenic microorganisms invade the blood circulation, multiply rapidly beyond the body's clearance capacity, and release toxins and metabolites, causing a series of serious clinical symptoms.

[0003] BSI has a high mortality rate, prolongs hospital stay, increases hospital costs, and is a serious threat. Timely identification of the etiology of BSI is the key to early treatment and improved prognosis.

[0004] In recent years, with the increase of invasive operations and the widespread use of broad-spectrum antibiotics, glucocorticoids and other drugs, the pathogen spectrum of BSI has changed significantly, and the proportion of Candida species has increased year by year. Among them, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei are the five most common Candida species causing Candida BSI, accounting for 95% of the total number of infections.

[0005] Therefore, establishing a method for rapid detection of Candida albicans from blood specimens, covering the above five common species, is an important issue that needs to be urgently addressed in clinical laboratories.

[0006] Currently, the gold standard for diagnosing Candida albicans BSI is still based on isolation and identification of blood culture, but the turnaround time (TAT) of this method is long, which is not conducive to early diagnosis and intervention. Although serological methods such as the G test can complete the test relatively quickly, their accuracy is low, false positives are prone to occur, and the infecting bacteria cannot be determined.

[0007] The above problems can be effectively solved by using methods based on polymerase chain reaction (PCR). It has high sensitivity and strong specificity, and can quickly detect pathogenic bacteria from blood samples. The method includes PCR-single-strand conformation polymorphism analysis, PCR-restriction fragment length polymorphism analysis, PCR-sequencing analysis, PCR-gene chip analysis, and real-time PCR. The first four methods require subsequent operations after PCR to complete the identification, while real-time PCR can make identifications at the same time during the PCR process, thereby further shortening TAT.

[0008] Research on the detection of microorganisms based on PCR methods has focused on ribosomal RNA (rRNA) encoding genes and used them as targets for identification. rRNA is a type of ribonucleic acid in cells that is widely present in prokaryotes and eukaryotes. The gene encoding rRNA is called ribosomal DNA (rDNA). The rRNA of eukaryotic organisms such as fungi can be divided into 18S rRNA, 5.8S rRNA and 28S rRNA according to the sedimentation coefficient. The corresponding 18S rDNA, 5.8S rDNA and 28S rDNA are also arranged in this order on the DNA. Among them, the Candida 5.8S rDNA sequence is highly conserved within the genus and can be used to design detection primers and probes for the Candida genus. Summary of the invention

[0009] In summary, the present invention aims to provide a method and a kit with significantly improved detection efficiency and accuracy, so as to quickly and accurately detect five common Candida species in BSI, including Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei.

[0010] Specifically, the present invention provides a set of amplification primers and Taqman-MGB probes based on the Candida 5.8S rDNA sequence for use in the implementation of real-time PCR detection.

[0011] Furthermore, the sequences of the amplification primers and Taqman-MGB probe are as follows:

[0012] The present invention also provides a kit for rapidly detecting common Candida species in BSI from a whole blood sample, wherein the kit comprises the above primers and a Taqman-MGB probe.

[0013] Furthermore, the kit comprises a nucleic acid amplification reagent and double distilled water without DNase. The working concentrations of the nucleic acid amplification reagent components are as follows: Heat-modified DNA polymerase 0.3U <![CDATA[MgCl2]]> 0.6mmol / L Tris-HCl (pH = 8.0) 20mmol / L KCl 50mmol / L dNTPs 1mmol / L

[0014] Furthermore, in the above-mentioned nucleic acid amplification reagent, each component is mixed in a concentrated manner, and the concentration is twice the working concentration. During use, a portion of the nucleic acid amplification reagent is added with primers and Taqman-MGB probes and then made up to an equal volume with double distilled water.

[0015] Furthermore, the kit comprises red blood cell lysing solution and white blood cell lysing solution. The components of the red blood cell lysing solution and the white blood cell lysing solution are as follows:

[0016] Furthermore, the kit may also include a calibrator, a positive control and a negative control. Preferably, the calibrator is a 10 7 CFU / mL-10 1 The positive control is a mixture of DNA of Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei in equal proportions. The negative control is sterile double distilled water without DNase.

[0017] The present invention also provides a method for rapidly detecting Candida from a whole blood sample, which is based on the above-mentioned kit including PCR amplification primers and Taqman-MGB probes to complete the detection.

[0018] In some embodiments, after red blood cells and white blood cells are removed by a lysis solution, the Candida DNA in the sample is released by a heat shock method; amplification is performed on a fluorescent quantitative PCR instrument based on the nucleic acid amplification reagent, PCR amplification primers and Taqman-MGB probe, and the S-shaped curve of the real-time fluorescence amplification curve is used to determine whether the blood sample contains Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei.

[0019] Furthermore, the amplification system includes: 10 μL of nucleic acid amplification reagent, 2 μL of sample DNA, 2 μL of upstream and downstream amplification primers (0.5 μmol / L), 1 μL of Taqman-MGB probe (0.25 μmol / L), and water to make up to 20 μL.

[0020] The amplification conditions may be:

[0021] It can be understood that in the above-mentioned Candida detection method, the detected Candida can be derived from the internal or external environment of the body; preferably, the external environment of the body includes Candida in various external environments, such as Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei; and the internal environment of the body can be derived from various tissues in the body, preferably, from peripheral whole blood specimens, such as Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei.

[0022] Compared with the prior art, the present invention has at least the following advantages:

[0023] (1) The present invention provides a rapid detection kit, which includes real-time PCR technology based on Taqman-MGB probe, and is applied for the first time to the detection of Candida albicans in whole blood specimens. The detection can be completed within 6 hours, thereby shortening the TAT and facilitating the early diagnosis and timely treatment of the disease.

[0024] (2) The present invention obtains a detection system suitable for common Candida species through sequence screening and optimization of primers and probes. Five common Candida species in BSI are detected based on this system. Combined with fluorescent quantitative PCR, the amplification results can be identified in real time, avoiding complex traditional detection methods and improving the efficiency of detection.

[0025] (3) The detection limit of the present invention for Candida albicans, Candida parapsilosis, Candida tropicalis and Candida krusei in whole blood specimens is 10 1 CFU / mL, the detection limit of Candida glabrata in whole blood specimens is 10 2 CFU / mL.

[0026] (4) The primers and Taqman-MGB probe designed in the present invention do not cross-react with non-Candida pathogens such as bacteria and human DNA, showing good specificity.

[0027] (5) The present invention is simple to operate and suitable for industrial promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Amplification curve and standard curve of primers and probe sets for preparation of gradient pure bacterial solution of Candida albicans standard strain.

[0029] Figure 2 Amplification curve and standard curve of primers and probe sets for preparation of gradient pure bacterial solution of Candida glabrata standard strain.

[0030] Figure 3 Amplification curve and standard curve of primers and probe sets for preparation of gradient pure bacterial solution of Candida parapsilosis standard strain.

[0031] Figure 4 Amplification curve and standard curve of primers and probe sets for preparation of gradient pure bacterial solution of Candida tropicalis standard strain.

[0032] Figure 5 Amplification curve and standard curve of primers and probe sets for preparation of gradient pure bacterial solution of Candida krusei standard strain.

[0033] Figure 6 Amplification curve and standard curve of the Candida albicans standard strain preparation gradient simulated infection blood specimen kit.

[0034] Figure 7 Amplification curve and standard curve of the gradient simulated infection blood specimen preparation kit for Candida glabrata standard strain.

[0035] Figure 8 Amplification curve and standard curve of the gradient simulated infection blood specimen preparation kit of Candida parapsilosis standard strain.

[0036] Fig. 9 Amplification curve and standard curve of the gradient simulated infection blood specimen preparation kit for Candida tropicalis standard strain.

[0037] Fig.10 Amplification curve and standard curve of the gradient simulated infection blood specimen preparation kit for Candida krusei standard strain. DETAILED DESCRIPTION

[0038] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if no specific conditions are specified, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased from the market.

[0039] Unless otherwise defined below, the meaning of all technical terms and scientific terms used in the specific embodiments of the present invention are intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain this product.

[0040] As used in the present invention, the terms "comprising", "including", "having", "containing" or "involving" are inclusive or open-ended, and do not exclude other unrecited elements or method steps. The term "consisting of..." is considered to be a preferred embodiment of the term "comprising".

[0041] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" is used, this includes a plural of that noun.

[0042] The terms “approximately” and “substantially” in the present invention represent an accuracy range that can be understood by those skilled in the art and can still ensure the technical effect of the feature in question.

[0043] Example 1: Design of primer / probe system.

[0044] (1) Download the 5.8S rDNA sequence of the Candida albicans standard strain from the “nucleotide” database of PubMed (http: / / www.ncbi.nlm.nih.gov / pubmed / ).

[0045] (2) Use the BLAST software in PubMed, enter the target sequence in "Enter Query Sequence"; select the "nucleotide collection" database in "Database" of "Choose Search Set" and the comparison database in "Organism"; select "Highly similar sequences (megablast)" in "Program Selection"; and click the "BLAST" button for comparison.

[0046] (3) Specific comparison steps:

[0047] 1) Compare the 5.8S rDNA sequences of Candida albicans and select highly conservative sequences with no base variation or low variation rate within the genus.

[0048] 2) The screened highly conserved sequences are compared with the human genome library (Homo), bacterial library (Bacter), fungal library (Fungi), virus library (Virus), Chlamydia library (Chlamydia), mycoplasma library (Mycoplasma), and Rickettsia library (Rickettsia) to determine whether there is a cross reaction.

[0049] 3) Primer and probe design: Primer and probe design was performed using Primer Premier 5.0 software based on the highly conserved sequences screened after alignment.

[0050] 4) Use the primer-BLAST software in PubMed to theoretically evaluate the detection rate of the designed primers and probes. The theoretical detection rate is calculated according to the following formula: theoretical detection rate = total number of Candida albicans strains in the database - number of strains that the primers and probes cannot correctly match) / total number of Candida albicans strains in the database × 100%.

[0051] (4) Theoretical detection rate of Candida primers and probes:

[0052] The theoretical detection sensitivity rate was calculated based on the above-mentioned variations. The detection rates for Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei were 90.4%, 97.9%, 86.7% and 98.0%, respectively. See the table below for details.

[0053] The screened primers were compared with the human genome library (Homo), bacterial library (Bacter), fungal library (Fungi), viral library (Virus), Chlamydia library (Chlamydia), mycoplasma library (Mycoplasma), and Rickettsia library (Rickettsia) by Primer-BLAST, showing no cross reaction (nonspecific amplification).

[0054] Example 2: Candida albicans count, preparation of gradient dilution standard bacterial solution and extraction of its DNA.

[0055] (1) The standard strain of Candida albicans was inoculated on Sabouraud medium and cultured in a CO2 incubator at 25°C for 48 hours.

[0056] (2) Pick several isolated Candida colonies and place them in 1 mL of sterile phosphate-buffered saline (PBS) buffer.

[0057] (3) After mixing the bacterial solution by pipetting, aspirate 100 μL and place it in 900 μL PBS for 10-fold dilution.

[0058] (4) Clean the modified abalone blood cell counting chamber, dry it, and cover it with a clean coverslip. Pipette 10 μL of the diluent and place it in the counting chamber. After standing for a few minutes, count the number of spores in the large squares on the four corners under a microscope. The counting principle is "count the top but not the bottom, count the left but not the right". When counting the budding spores, if the bud reaches half the size of the mother cell, it can be counted as two bacteria; if the bud is smaller than half the size of the mother cell, it is still counted as one yeast cell.

[0059] (5) Formula conversion: Number of Candida spores per ml = (N / 4) × 10 × 10 × 10 3 (CFU). In the formula, "N" is the total number of spores in the four large squares, "÷4" is the average number of white blood cells in each large square (i.e. 0.1μl), "×10" is the volume of one large square of 0.1μL, converted to 1.0μL, "×20" is the dilution multiple of the bacterial solution, and "×10 3 ” is 1 μL converted to 1 mL.

[0060] (6) According to the Candida count results, the original bacterial solution was prepared to a concentration of 10 7 CFU / mL standard bacterial solution. Continuously dilute 10 times to obtain a concentration of 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 1 CFU / mL and 10 0CFU / mL gradient dilution standard bacterial solution.

[0061] (7) The bacterial solution was heated at 100°C for 15 minutes, and then frozen at -80°C for 10 minutes. This was repeated three times, and then centrifuged at 12,000 r / min for 15 minutes. The supernatant was the crude Candida DNA extract, which was aspirated and stored at -80°C for subsequent verification of detection sensitivity and as a calibration solution.

[0062] Example 3: Preparation of simulated Candida infection blood specimens and extraction of DNA

[0063] (1) Prepare a gradient dilution of the Candida albicans standard solution according to the above method, with a concentration range of 10 7 -10 1 CFU / mL.

[0064] (2) Take 100 μL of each concentration and inject it into 900 μL of EDTA anticoagulated whole blood sample from healthy volunteers to prepare gradient simulated infected blood sample with a concentration range of 10 6 -10 0 CFU / mL.

[0065] (3) Add 500 μL of red blood cell lysis buffer to the gradient simulated infected blood sample, incubate at room temperature for 10 min, centrifuge at 12,000 rpm for 15 min, and discard the supernatant.

[0066] (4) Add 1 mL of leukocyte lysis buffer to the precipitate, incubate at room temperature for 5 min, centrifuge at 12,000 rpm for 15 min, and discard the supernatant.

[0067] (5) Add 400 μL PBS to resuspend the pellet and extract Candida DNA according to the method described above.

[0068] Example 4: Verification of detection sensitivity.

[0069] The Candida test kit was used to test the gradient dilutions of five Candida species. The results showed that the detection limit of the kit for the five Candida species was 10 1 CFU / mL( Figure 1-Figure 5 ). Figure 1 Preparation of gradient pure bacterial solution PCR amplification curve and standard curve for Candida albicans standard strain, A: PCR amplification curve; B: PCR standard curve. 6-1 in the figure represents a concentration of 10 6 -10 1 CFU / mL of pure Candida albicans solution. Figure 2 The PCR amplification curve and standard curve of the gradient pure bacterial solution prepared for the standard strain of Candida glabrata, A: PCR amplification curve; B: PCR standard curve, in the figure, 6-1 represents the concentration of 10 6 -101 CFU / mL of pure Candida glabrata solution. Figure 3 The PCR amplification curve and standard curve of the gradient pure bacterial solution prepared for the standard strain of Candida parapsilosis, A: PCR amplification curve; B: PCR standard curve, in the figure, 6-1 represents the concentration of 10 6 -10 1 CFU / mL of pure Candida parapsilosis culture fluid. Figure 4 Preparation of gradient pure bacterial solution PCR amplification curve and standard curve for Candida tropicalis standard strain, A: PCR amplification curve; B: PCR standard curve, 6-1 in the figure represents the concentration of 10 6 -10 1 CFU / mL of pure Candida tropicalis liquid. Figure 5 The PCR amplification curve and standard curve of the gradient pure bacterial solution prepared for the standard strain of Candida krusei, A: PCR amplification curve; B: PCR standard curve, in the figure, 6-1 represents the concentration of 10 6 -10 1 CFU / mL of pure Candida krusei culture liquid. The Candida test kit was used to test the simulated infection blood samples prepared with gradient dilutions of five Candida species four times. 6 -10 2 CFU / mL level, the four test results of five Candida species were all positive ( Figure 6-Figure 10 ). Figure 6 Amplification curve and standard curve of gradient simulated infection blood specimen kit for Candida albicans standard strain preparation, A: PCR amplification curve; B: PCR standard curve, 6-1 in the figure represents the concentration of 10 6 -10 1 CFU / ml of Candida albicans simulated infection blood specimens. Figure 7 Amplification curve and standard curve of gradient simulated infection blood specimen kit for Candida glabrata standard strain preparation, A: PCR amplification curve; B: PCR standard curve, 6-1 in the figure represents the concentration of 10 6 -10 1 CFU / ml of Candida glabrata simulated infection blood specimens. Figure 8 Amplification curve and standard curve of gradient simulated infection blood specimen kit for Candida parapsilosis standard strain preparation, A: PCR amplification curve; B: PCR standard curve, 6-1 in the figure represents the concentration of 10 6 -10 1 CFU / ml of Candida parapsilosis simulated infection blood specimens. Fig. 9Amplification curve and standard curve of gradient simulated infection blood specimen kit for preparation of standard strain of Candida tropicalis. A: PCR amplification curve; B: PCR standard curve. In the figure, 6-1 represents the concentration of 10 6 -10 1 CFU / ml of Candida tropicalis simulated infection blood specimens. Fig.10 Amplification curve and standard curve of gradient simulated infection blood specimen kit for Candida krusei standard strain preparation, A: PCR amplification curve; B: PCR standard curve, 6-1 in the figure represents the concentration of 10 6 -10 1 CFU / ml of Candida krusei simulated infection blood specimens.

[0070] At a concentration of 10 1 CFU / mL level, the four tests for Candida albicans, Candida parapsilosis, Candida tropicalis and Candida krusei were all positive ( Figure 6 , Figure 8-Figure 10 ), while in the 4 tests for Candida glabrata, 3 were positive ( Figure 7 ).

[0071] Example 5: Precision verification.

[0072] In the intra-batch test of simulated blood samples, the maximum CV values ​​appeared in 10 of the five standard strains. 1 The CFU / mL levels ranged from 0.96% to 1.22%. The CV values ​​varied significantly between batches, ranging from 10 6 CFU / mL levels ranged from 0.36% to 10 1 The CFU / ml level was 2.22%, see the table below for details. Table of CV values ​​(%) of intra-batch test of Candida species detection kit CFU / mL C.albicans C. glabrata C.parapsilosis C. tropicalis C. krusei <![CDATA[10 6 ]]> 0.49 0.09 0.18 0.24 0.21 <![CDATA[10 5 ]]> 0.49 0.72 0.12 0.13 0.58 <![CDATA[10 4 ]]> 0.40 0.27 0.25 0.32 0.31 <![CDATA[10 3 ]]> 0.07 0.54 0.30 0.20 0.28 <![CDATA[10 2 ]]> 0.50 0.81 0.37 0.57 0.27 <![CDATA[10 1 ]]> 1.22 1.04 1.03 1.06 0.96 Table of CV values ​​(%) of Candida detection kit batch test

[0073] Example 5: Verification of detection specificity.

[0074] (1) This kit has no cross-reaction to common bacteria that cause BSI, such as Klebsiella pneumoniae, Escherichia coli, Proteus, Serratia marcescens, Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus.

[0075] (2) This kit has no cross-reaction with human genomic DNA.

[0076] In summary, the kit for detecting Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei provided by the present invention has the characteristics of high sensitivity, strong specificity and good stability, and can quickly and accurately detect the presence of Candida in blood specimens.

[0077] Example 6: Clinical application evaluation.

[0078] The study included 82 patients with suspected Candida bloodstream infection, and a total of 328 clinical blood specimens were obtained. These clinical blood specimens were used as samples for prospective studies, and the test results were compared with the test results of blood culture. The extraction of Candida DNA from blood specimens of suspected infected patients was carried out according to the extraction process described above. Through blood culture detection, 7 blood specimens were positive (tropical Candida) from 1 patient. Through the Candida PCR detection kit, 12 blood specimens were positive from 4 patients, including 7 blood specimens from patients with positive blood culture (all tropical Candida). For the remaining 316 blood specimens, the results of blood culture and PCR were all negative. Therefore, the sensitivity of this detection platform is 100% and the specificity is 98.4%, as shown in the table below. Comparison of diagnostic results between blood culture and PCR test

[0079] In this study, 5 patients had negative blood culture results but positive PCR results. After detailed inquiry of the case data, it was found that they were all given antifungal treatment before blood was drawn. Therefore, Candida in their blood samples was not easy to detect by blood culture, which further demonstrated the superiority of the detection method based on this kit.

[0080] The above embodiments are only preferred implementation modes of the present invention, and therefore cannot be used to limit the scope of implementation of the present invention. Without departing from the spirit and principle of the present invention, any changes, modifications, substitutions and combinations made are within the protection scope of the present invention.

Claims

1. A primer probe for rapid detection of common Candida species in bloodstream infection, characterized in that: The common Candida species are Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei; the sequences of the primers are: upstream primer: 5'-GGATCTCTTGGTTCTCGCATC-3', downstream primer: 5'-AACGACGCTCAAACAGGCAT-3', and the sequence of the probe is: FAM-CGCAATGTGCGTTCAA-MGB-NFQ.

2. A rapid detection kit for common Candida species in bloodstream infections, characterized in that: The kit comprises the primers and probes according to claim 1.

3. The rapid detection kit for common Candida fungi in bloodstream infection according to claim 2, characterized in that: The kit comprises a nucleic acid amplification reagent, wherein the nucleic acid amplification reagent comprises a DNA polymerase and a PCR buffer.

4. The rapid detection kit for common Candida fungi in bloodstream infection according to claim 3, characterized in that: The nucleic acid amplification reagent comprises: DNA polymerase 0.3U MgCl20.6mmol / L Tris-HCl, pH = 8.0 20mmol / L KCl 50mmol / L dNTPs 1mmol / L.

5. The rapid detection kit for common Candida species in bloodstream infection according to claim 3 or 4, characterized in that: The kit also includes a red blood cell lysing solution and a white blood cell lysing solution, and the lysing solution includes the following concentration components: Red blood cell lysis buffer: NH4Cl 8.3g / L KHCO31.0g / L EDTA-Na2 0.037 g / L; Leukocyte Lysis Buffer: Na2CO3 500mmol / L SDS10g / L.

6. The rapid detection kit for common Candida species in bloodstream infection according to any one of claims 2 to 4, characterized in that: The kit also includes a calibrator, a positive control and a negative control. The calibrator has a concentration of 10 7 CFU / mL-10 1 CFU / mL of Candida albicans suspension extracted DNA; the positive control is a mixture of DNA of Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei in equal proportions; the negative control is sterile DNase-free double distilled water.

7. Use of the primer probe according to claim 1 in preparing a kit for detecting common Candida species in bloodstream infection.

8. The application according to claim 7, characterized in that: The detection method of the kit comprises: using red blood cell lysis solution and white blood cell lysis solution to lyse red blood cells and white blood cells, releasing Candida DNA in the sample, and performing PCR amplification using the primers, probes and nucleic acid amplification reagents.

9. The use according to claim 8, characterized in that: The PCR amplification conditions are: 95° C. for 2 min; 95° C. for 15 s, 60° C. for 1 min, and 40 cycles.

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