Freeze-dried PCR reagent and kit for detecting invasive pulmonary candida nucleic acid

Abstract

The application discloses an invasive lung Candida nucleic acid detection freeze-dried PCR reagent and kit, which comprises ATG reaction freeze-dried powder, ATG positive control and ATG negative control; the ATG reaction freeze-dried powder comprises PCR Buffer, Taq DNA polymerase, dNTP, a freeze-drying protective agent, specific amplification Candida 5.8s rRNA-ITS2-25s rRNA gene oligonucleotide upstream primers and oligonucleotide downstream primers, specific detection of Candida albicans oligonucleotide fluorescent probes, specific detection of Candida tropicalis oligonucleotide fluorescent probes, specific detection of Candida glabrata oligonucleotide fluorescent probes, specific amplification of human genome beta-actin gene oligonucleotide upstream primers and oligonucleotide downstream primers and oligonucleotide fluorescent probes, which can not only avoid mutual interference between multiple pairs of primers, but also can identify and separate Candida albicans, Candida tropicalis and Candida glabrata, has high specificity and strong applicability, and can well assist Candida diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a lyophilized PCR reagent and kit for the detection of nucleic acid in invasive Candida albicans. Background Technology

[0002] Candida species are widespread in nature and are the most common opportunistic or conditionally pathogenic fungi, causing diseases that rank first among invasive fungal diseases (IFD). Clinically, Candida albicans is the most common, but there are more than 16 non-Candida species, among which Candida tropicalis and Candida glabrata are relatively common. Candidiasis is more common in immunocompromised patients and can invade local skin, mucous membranes, and various tissues and organs throughout the body, with diverse clinical manifestations and varying degrees of severity. In recent years, with the increase in risk factors such as tumor chemotherapy, organ transplantation, glucocorticoids, immunosuppressants, and broad-spectrum antibiotics, the incidence of invasive candiddiasis has shown a significant upward trend. Early diagnosis of candidiasis, especially invasive candidiasis, is often difficult, leading to delays in antifungal treatment and affecting patient prognosis.

[0003] Currently, commonly used clinical diagnostic methods include: direct microscopic examination of fungi, fungal culture and identification, fungal serological testing, and histopathological examination. Each of these methods has certain limitations: direct microscopic examination of fungi is simple to perform and provides rapid results, but suffers from false negatives and low sensitivity, and relies heavily on the experience of the laboratory personnel, making it difficult to distinguish the origin of the fungal flora; fungal culture is an important diagnostic method for fungal infections, valuable for diagnosing infection and identifying the pathogenic fungal species, but it is time-consuming, has a low positive rate and low sensitivity, and struggles to differentiate between colonization and infection; serological antigen testing is suitable for the early diagnosis of invasive fungal diseases, is quick, and provides rapid results, serving as a valuable supplement to traditional mycological examinations, but the results are influenced by many factors, have poor repeatability, and only have corresponding methods for a few fungi; histopathological examination is definitive evidence for invasive fungal diseases, but it is difficult to identify the fungal species, involves invasive sampling, and requires the assistance of other methods.

[0004] Molecular biology methods can detect very low levels of fungal DNA in a short time, exhibiting high sensitivity and specificity, which is beneficial for the early diagnosis of fungal infections. Multiplex fluorescent PCR is highly automated, eliminating the need for electrophoresis and hybridization, effectively reducing misinterpretations caused by contamination. Furthermore, due to its highly sensitive fluorescence changes, it is more sensitive than traditional PCR. In addition, it is simple to operate, requiring only one step to obtain results, and is convenient, rapid, and accurate.

[0005] Currently, multiplex quantitative real-time PCR (qPCR) is available for detecting Candida. For example, Chinese patent application CN201711395514 discloses a primer-probe combination and kit for simultaneously detecting four Candida species. This method uses one primer pair and one probe to detect Candida genus (Candida albicans, Candida tropicalis, Candida glabrata, and Candida parapsilosis), but it cannot distinguish between Candida species, which is not conducive to targeted drug treatment for Candida species in the later stages. For instance, Chinese patent application CN202311854288 discloses a fluorescent probe combination, detection kit, and application for detecting fungi in sputum. Each Candida species is detected using one primer pair and one probe. Although it can distinguish Candida species, the presence of multiple primer pairs in a single reaction system can lead to primer interference, affecting the accuracy of the detection results. Furthermore, all of the above methods use liquid reagents containing temperature-sensitive Taq enzymes, primers, and probes, requiring low-temperature transportation and storage to prevent changes in their properties and subsequent inactivation. The preparation process is also cumbersome, and the storage and transportation costs are high. Summary of the Invention

[0006] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a lyophilized PCR reagent and kit for detecting invasive Candida albicans nucleic acid, comprising: ATG reaction lyophilized powder, ATG positive control and ATG negative control; wherein the ATG reaction lyophilized powder comprises PCR buffer, Taq DNA polymerase, dNTP, lyophilization protectant, oligonucleotide upstream primer and oligonucleotide downstream primer for specifically amplifying the 5.8S rRNA-ITS2-25S rRNA gene of Candida spp., oligonucleotide fluorescent probe for specifically detecting Candida albicans, oligonucleotide fluorescent probe for specifically detecting Candida tropicalis, oligonucleotide fluorescent probe for specifically detecting Candida glabrata, and oligonucleotide upstream primer and oligonucleotide downstream primer and oligonucleotide fluorescent probe for specifically amplifying the β-actin gene on the human genome.

[0007] According to the present invention, the lyophilized PCR reagent and kit for detecting invasive Candida albicans nucleic acid is provided, wherein the oligonucleotide fluorescent probe is labeled with a fluorescent group and a quencher group at both ends, the fluorescent group being one of FAM, VIC, CY5, and ROX, and the quencher group being one of BHQ1, BHQ2, BHQ3, TAMRA, and MGB.

[0008] According to the present invention, a lyophilized PCR reagent and kit for detecting invasive Candida albicans nucleic acid comprises, in each tube of ATG reaction lyophilized powder: 0.1 μM-1 μM oligonucleotide upstream primer, 0.1 μM-1 μM oligonucleotide downstream primer, 0.1 μM-1 μM oligonucleotide fluorescent probe, 0.1-1 unit of Taq DNA polymerase, lyophilization protectant at 1%-10% m / v, 0.1-1 mM dNTP, and 1-10× PCR buffer.

[0009] According to the present invention, a lyophilized PCR reagent and kit for detecting invasive Candida albicans nucleic acid comprises, in each tube of ATG reaction lyophilized powder: 0.6 μM Candida spp. oligonucleotide upstream and downstream primers, 0.2 μM Candida albicans oligonucleotide fluorescent probes and Candida tropicalis oligonucleotide fluorescent probes, 0.4 μM Candida glabrata oligonucleotide fluorescent probes, 0.2 μM β-actin gene oligonucleotide upstream and downstream primers, 0.1 μM β-actin gene oligonucleotide fluorescent probes, 0.8 units of Taq DNA polymerase, lyophilization protectant with a m / v of 2.5%, 0.6 mM dNTP, and 5× PCR buffer.

[0010] According to the present invention, the lyophilized PCR reagent and kit for detecting invasive pulmonary Candida nucleic acid, wherein the Taq DNA polymerase is a mutant Taq DNA polymerase, and the enzyme is one of chemical modification, antibody modification, or ligand modification.

[0011] According to the present invention, the lyophilized PCR reagent and kit for detecting invasive Candida albicans nucleic acid uses a lyophilization protectant that is one or more of the following: glycine, trehalose, sucrose, mannitol, gelatin, PEG-20000, hydroxypropyl-β-cyclodextrin, PVP-10000, maltodextrin, sodium carboxymethyl cellulose, etc.

[0012] According to the present invention, a lyophilized PCR reagent and kit for detecting invasive pulmonary Candida nucleic acid, wherein the ATG positive control comprises a plasmid containing a Candida albicans target gene sequence, a plasmid containing a Candida tropicalis target gene sequence, a plasmid containing a Candida glabrata target gene sequence, and a plasmid containing a specific fragment of the human β-actin gene, wherein the concentration of each target gene plasmid is 10. 5The human β-actin gene fragment plasmid concentration was 10 copies / mL. 6 copies / mL.

[0013] According to the present invention, a lyophilized PCR reagent and kit for detecting invasive pulmonary Candida nucleic acid is provided, wherein the ATG positive control is a plasmid containing a specific fragment of the human β-actin gene, and the plasmid concentration is 10. 6 copies / mL.

[0014] According to the present invention, a lyophilized PCR reagent and kit for detecting invasive Candida albicans nucleic acid is provided, wherein the kit comprises: the reagent composition according to claim 1.

[0015] Beneficial effects:

[0016] (1) This invention designs a pair of common detection primers on the conserved regions of 5.8S rRNA and 25S rRNA in the Candida genome. It uses the different sequences on the diverse endogenous transcription spacer (ITS) to distinguish Candida albicans, Candida tropicalis and Candida glabrata. This avoids the mutual interference between multiple primer pairs and can identify and separate Candida albicans, Candida tropicalis and Candida glabrata. It has high specificity and strong applicability and can greatly assist in the diagnosis and treatment of Candida.

[0017] (2) The present invention uses a mutant antibody to modify Taq DNA polymerase. This enzyme can rapidly release its activity at 95°C, which can greatly shorten the detection time. The detection can be completed in about 55 minutes.

[0018] (3) The ATG reaction lyophilized powder prepared by the present invention comprises oligonucleotide primers and probes of each target and internal standard, Taq DNA polymerase, dNTP, lyophilization protectant, PCR buffer and other components, which are prepared by lyophilization into components that are stable and coexist at room temperature. The detection performance is consistent with that of liquid reagents. It greatly reduces the transportation and storage costs of fluorescent PCR reagents, increases the flexibility of sample volume, improves detection sensitivity, reduces reagent preparation steps, simplifies the clinical testing process, and is more convenient for practical use and promotion. Attached Figure Description

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0020] Figure 1 This is an example diagram of the ATG reaction pair dry powder of the lyophilized PCR reagent and kit for detecting invasive Candida albicans nucleic acid according to the present invention;

[0021] Figure 2 This is a comparison chart of the detection performance of the ATG reaction lyophilized powder and liquid PCR reagent of the lyophilized PCR reagent and kit for detecting invasive Candida albicans nucleic acid of the present invention.

[0022] Figure 3 The figure shows the stability test results of the lyophilized PCR reagent and kit for detecting invasive Candida albicans nucleic acid of the present invention.

[0023] Figure 4 This is an example of clinical sample detection results for the lyophilized PCR reagent and kit for detecting invasive pulmonary Candida nucleic acid according to the present invention. Detailed Implementation

[0024] This section will describe in detail specific embodiments of the present invention. Preferred embodiments of the present invention are shown in the accompanying drawings. The purpose of the drawings is to supplement the textual description with graphics, so that people can intuitively and vividly understand each technical feature and overall technical solution of the present invention, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] Primer and probe design for targets and internal standards

[0027] The 5.8S rRNA-ITS2-25S rRNA gene sequences of Candida albicans, Candida tropicalis, and Candida glabrata, as well as the human β-actin gene sequence, were searched on NCBI (National Center of Biotechnology Information). Specific primer and probe sequences were designed according to primer and probe design principles and synthesized by Jiangsu Kangwei Century Biotechnology Co., Ltd. After sequence synthesis, quality control and quantification were performed, followed by dilution to the required concentration as needed.

[0028] The primer and probe sequences of the target and internal standard used in this invention are shown in the table below.

[0029]

[0030] Example 2

[0031] Preparation of lyophilized PCR reagents and kits for the detection of invasive pulmonary Candida nucleic acid (48 doses / kit)

[0032] (1) ATG Reactive Lyophilized Powder

[0033] Prepare a 25% (m / v) freeze-drying protectant stock solution: 25% maltodextrin + 25% trehalose + 25% gelatin + 25% mannitol. Here, m / v refers to mass concentration, indicating the mass per unit volume, expressed in kg / L or kg / m³.

[0034] The ATG reactive lyophilized powder is prepared as follows:

[0035]

[0036]

[0037] After thoroughly mixing the above components, a final reaction volume of 25 μL of ATG reaction lyophilized powder is obtained. Prepare the required volume of mixture (25 μL × N) as needed, mix thoroughly, and aliquot into 8-tube PCR tubes. Place the tubes uncapped in a lyophilizer to prepare the ATG reaction lyophilized powder, following the following procedure: 0℃, 30 min; -45℃, 4 h; -30℃, 14 bar, 3 h; -10℃, 14 bar, 2.5 h; 0℃, 14 bar, 2 h; 30℃, 14 bar, 4 h. After lyophilization, cap the PCR tubes and vacuum seal them in aluminum foil bags (with desiccant inside) to obtain the ATG reaction lyophilized powder.

[0038] (2) ATG positive control: plasmids containing the target gene sequence of Candida albicans, plasmids containing the target gene sequence of Candida tropicalis, plasmids containing the target gene sequence of Candida glabrata, and plasmids containing a specific fragment of the human β-actin gene. The concentration of the target gene plasmids was 10. 5 copies / mL; human β-actin gene fragment plasmid concentration was 10. 6 copies / mL.

[0039] (3) Negative control: Plasmid containing a specific fragment of the human β-actin gene, with a plasmid concentration of 10. 6 copies / mL.

[0040] The kit (48 doses / box) components are shown in the table below:

[0041]

[0042]

[0043] Example 3

[0044] Detection Procedure for Lyophilized PCR Reagents and Kits for the Detection of Invasive Pulmonary Candida albicans Nucleic Acid

[0045] 1. Sample pretreatment

[0046] 1.1 Sputum

[0047] Take 200 μL of sample, add 2 times the volume of 4% NaOH, and incubate at 37℃ and 600 rpm for 15–30 min to fully liquefy the sputum, and use it as the sample to be tested.

[0048] 1.2 Bronchoalveolar lavage fluid

[0049] Clarified sample: No liquefaction is required; it can be used directly as the sample to be tested.

[0050] For samples containing a small amount of viscous sputum: Centrifuge as much bronchoalveolar lavage fluid as possible, and collect the lower viscous portion. Then, liquefy the viscous portion as you would a sputum sample and use it as the sample to be tested.

[0051] 2. Sample processing

[0052] Take 400 μL of the sample to be tested, ATG positive control, and ATG negative control. It is recommended to use the nucleic acid extraction reagent (Su Tai Medical Device Registration No. 20250032) produced by Jiangsu Kangwei Century Biotechnology Co., Ltd., and perform nucleic acid extraction according to its instructions.

[0053] 3. Sample addition

[0054] 3.1 Preparation of reaction solution: Calculate the required number of reactions based on the number of samples to be tested. If the number of samples is n, then the total number of reactions N = n + 2. Take N tubes of ATG lyophilized reaction powder, centrifuge for 30 seconds to ensure that the ACP lyophilized reaction powder reaches the bottom of the tube, and set aside for later use.

[0055] 3.2 Sample addition: Gently open the cap of the eight-tube containing the lyophilized powder, transfer 25 μL of the extracted test sample, ATG positive control and ATG negative control nucleic acid into the eight-tube, and tighten the cap.

[0056] 3.3 Vortex for 30 seconds until completely dissolved and the solution is clear with no white lyophilized residue. Remove air bubbles and centrifuge briefly for 30 seconds. Set aside for later use.

[0057] 4. PCR amplification: This invention uses an ABI 7500 real-time quantitative PCR instrument for detection. The pre-denaturation process is performed at 95℃ for 30 seconds, followed by denaturation at 95℃ for 10 seconds, and annealing and extension at 58℃ for 30 seconds. This process is repeated for 40 cycles. Candida albicans is detected in the ROX channel, Candida tropicalis in the FAM channel, Candida glabrata in the CY5 channel, and human β-actin gene in the VIC channel.

[0058] 5 Results Analysis

[0059] 5.1 Quality Control Standards

[0060] ATG positive control: The Ct value of the target corresponding to the FAM, ROX, VIC and CY5 channels should be ≤35;

[0061] ATG negative control: FAM, ROX and CY5 channels showed no amplification curve (No Ct) or Ct value > 38, and the corresponding target Ct value of VIC channel should be ≤ 35;

[0062] All of the above requirements must be met simultaneously in the same experiment; otherwise, the experiment will be invalid and the test must be repeated.

[0063] 5.2 Result Determination

[0064]

[0065] For samples that test positive for Candida albicans, Candida tropicalis, or Candida glabrata, the internal standard test result is not required. For negative samples, the internal standard test should be positive. If the internal standard test is negative, the test result of the sample is invalid. The cause should be found and eliminated, and the sample should be retested for repeated experiments.

[0066] Example 4

[0067] Comparison of detection performance between ATG reaction lyophilized powder and liquid PCR reagent

[0068] The ATG reaction lyophilized powder used in this embodiment is the ATG reaction lyophilized powder for the detection of invasive pulmonary Candida nucleic acid prepared in Example 2, and the control reagent is liquid PCR reagent (with the same formulation as the ATG reaction lyophilized powder).

[0069] ATG Reactive Lyophilized Powder: ATG reactive lyophilized powder + 10 μL of test sample + 15 μL of ddH2O

[0070] Liquid amplification system: Prepare the liquid amplification system according to the table below, vortex to mix, and dispense 15 μL / well.

[0071] Components Dosage per person (μL) Final concentration CA-F (100μM) 0.15 0.6μM CA-R (100μM) 0.15 0.6μM CA-PA (100μM) 0.05 0.2μM CA-PT (100μM) 0.05 0.2μM CA-PG (100μM) 0.1 0.4μM Actin-F (100μM) 0.05 0.2μM Actin-R (100μM) 0.05 0.2μM Actin-P (100μM) 0.025 0.1μM Lyophilization protectant stock solution (25%) 2.5 2.5％ Taq DNA polymerase (5 units) 4 0.8 units dNTPs (10mM) 1.5 0.6mM 5×PCRBuffer 5 1× <![CDATA[ddH2O]]> 1.375 / Total 15

[0072] Template: ATG positive control, ATG negative control and normal human sputum samples prepared in Example 2.

[0073] The detection method was the same as in Example 3. The results of the ATG positive control and ATG negative control met the quality control standards, and the test was valid.

[0074] ATG-reacting lyophilized powder, such as Figure 1 As shown, the test results are as follows Figure 2 As shown: The ACP reaction lyophilized powder prepared for the detection of invasive pulmonary Candida nucleic acid has the same detection performance as the liquid PCR reagent (with the same formulation as the ATG reaction lyophilized powder).

[0075] Example 5

[0076] Performance testing of lyophilized PCR reagents and kits for the detection of invasive pulmonary Candida nucleic acid.

[0077] 1. Detection limit

[0078] The templates consisted of five different concentrations of Candida albicans / Candida tropicalis / Candida glabrata cultures, with 25 μL of RNase-Free Water used as a template-free control (NTC). All templates were replicated 20 times. The kits prepared in Example 2 were used, and the detection method was the same as in Example 3.

[0079] The results are shown in the table below: the detection sensitivity for Candida albicans, Candida tropicalis, and Candida glabrata was 200 copies / mL.

[0080] Results of the detection limit test for invasive pulmonary Candida

[0081]

[0082] 2. Cross-reaction

[0083] DNA was extracted from *Candida glabrata*, *Candida krusei*, *Aspergillus fumigatus*, *Aspergillus niger*, *Aspergillus flavus*, *Aspergillus oryzae*, *Aspergillus terreus*, *Cryptococcus neoformans*, *Cryptococcus gutterus*, *Streptococcus pneumoniae*, *Escherichia coli*, and *Staphylococcus aureus* using the method described in Example 3. The detection method was the same as in Example 3, using the kit prepared in Example 2.

[0084] The results are shown in the table below: The kit provided by this invention does not produce cross-reactions with other Candida species (Candida parapsilosis, Candida krusei); it does not produce cross-reactions with other common clinical fungal pathogens (Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Aspergillus terreus, Cryptococcus neoformans, Cryptococcus gutterusii); in addition, there are no cross-reactions with Streptococcus pneumoniae, Escherichia coli and Staphylococcus aureus.

[0085]

[0086] Note: Undet means Undetermined.

[0087] 3. Stability

[0088] The kit used in this embodiment is the lyophilized PCR reagent and kit for detecting invasive pulmonary Candida nucleic acid prepared in Example 2. This kit was stored at room temperature for 12 months and compared with a newly prepared lyophilized PCR reagent and kit (prepared using the same method as in Example 2). The test samples were ATG positive and ATG negative controls, and the detection method was the same as in Example 3.

[0089] Test results are as follows Figure 3 As shown, the amplification performance of the lyophilized PCR kit stored at room temperature for 12 months was consistent with that of the newly prepared lyophilized PCR kit in the control group. Therefore, the lyophilized PCR reagent and kit (48 tests / kit) prepared in Example 2 for the detection of invasive Candida albicans nucleic acid can be stored at room temperature for 12 months.

[0090] Example 6

[0091] Application of lyophilized PCR reagents and kits for the detection of invasive pulmonary Candida in clinical samples

[0092] The reagents used were the lyophilized PCR reagents and kits (48 samples / kit) prepared in Example 2 for the detection of invasive pulmonary Candida nucleic acid. The samples to be tested were 2 Candida albicans positive sputum samples, 2 Candida tropicalis positive sputum samples, 2 Candida glabrata positive sputum samples, and 5 negative sputum samples remaining from the hospital's laboratory.

[0093] The detection method was the same as in Example 3. The results of the ATG positive and negative controls met the quality control standards, and the test was valid. The test results are shown in the table below: 2 samples were positive for Candida albicans ( Figure 4 Example); 2 samples were positive for Candida tropicalis ( Figure 4 Example); 2 samples were positive for Candida glabrata ( Figure 4 Example); 5 samples were negative ( Figure 4 Example); consistent with the hospital's diagnosis.

[0094]

[0095] Note: Undet means Undetermined.

[0096] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A lyophilized PCR reagent for detecting invasive pulmonary Candida nucleic acid, characterized in that, include: ATG reaction lyophilized powder, ATG positive control and ATG negative control; The ATG reaction lyophilized powder includes PCR buffer, Taq DNA polymerase, dNTPs, lyophilization protectant, oligonucleotide upstream and downstream primers for specifically amplifying the 5.8S rRNA-ITS2-25S rRNA gene of Candida spp., oligonucleotide fluorescent probes for specifically detecting Candida albicans, oligonucleotide fluorescent probes for specifically detecting Candida tropicalis, oligonucleotide fluorescent probes for specifically detecting Candida glabrata, and oligonucleotide upstream and downstream primers and oligonucleotide fluorescent probes for specifically amplifying the β-actin gene on the human genome.

2. The lyophilized PCR reagent for detecting invasive pulmonary Candida nucleic acid according to claim 1, characterized in that, The oligonucleotide fluorescent probe is labeled with a fluorescent group and a quencher group at both ends. The fluorescent group is one of FAM, VIC, CY5, and ROX, and the quencher group is one of BHQ1, BHQ2, BHQ3, TAMRA, and MGB.

3. The lyophilized PCR reagent for detecting invasive pulmonary Candida nucleic acid according to claim 1, characterized in that, Each tube of ATG reaction lyophilized powder contains: 0.1 μM-1 μM oligonucleotide upstream primer, 0.1 μM-1 μM oligonucleotide downstream primer, 0.1 μM-1 μM oligonucleotide fluorescent probe, 0.1-1 unit of Taq DNA polymerase, lyophilization protectant at 1%-10% m / v, 0.1-1 mM dNTP, and 1-10× PCR Buffer.

4. The lyophilized PCR reagent for detecting invasive pulmonary Candida nucleic acid according to claim 1, characterized in that, Each tube of ATG reaction lyophilized powder contains: 0.6 μM Candida spp. oligonucleotide upstream and downstream primers, 0.2 μM Candida albicans oligonucleotide fluorescent probes and Candida tropicalis oligonucleotide fluorescent probes, 0.4 μM Candida glabrata oligonucleotide fluorescent probes, 0.2 μM β-actin gene oligonucleotide upstream and downstream primers, 0.1 μM β-actin gene oligonucleotide fluorescent probes, 0.8 units of Taq DNA polymerase, lyophilization protectant at 2.5% m / v, 0.6 mM dNTP, and 5× PCR Buffer.

5. The lyophilized PCR reagent for detecting invasive pulmonary Candida nucleic acid according to claim 1, characterized in that, The Taq DNA polymerase is a mutant Taq DNA polymerase, which is modified by one of the following methods: chemical modification, antibody modification, or ligand modification.

6. The lyophilized PCR reagent for detecting invasive pulmonary Candida nucleic acid according to claim 1, characterized in that, The freeze-drying protectant is one or more of the following: glycine, trehalose, sucrose, mannitol, gelatin, PEG-20000, hydroxypropyl-β-cyclodextrin, PVP-10000, maltodextrin, sodium carboxymethyl cellulose, etc.

7. The lyophilized PCR reagent for detecting invasive pulmonary Candida nucleic acid according to claim 1, characterized in that, The ATG positive control consisted of a plasmid containing a Candida albicans target gene sequence, a plasmid containing a Candida tropicalis target gene sequence, a plasmid containing a Candida glabrata target gene sequence, and a plasmid containing a human β-actin gene-specific fragment. The concentration of the target gene plasmid was 10⁵ copies / mL, and the concentration of the human β-actin gene fragment plasmid was 10⁶ copies / mL.

8. A kit for detecting lyophilized PCR reagents for invasive pulmonary Candida nucleic acid, characterized in that, The kit comprises the reagent composition of claim 1.

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