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

Abstract

The application discloses a freeze-dried PCR reagent and kit for detecting invasive lung fungus nucleic acid, which comprises ACP reaction freeze-dried powder, ACP positive control and ACP negative control; the ACP reaction freeze-dried powder comprises PCR Buffer, dNTP Mix, Taq DNA Polymerase, freeze-dried protective agent and specific primers and probes; the PCR Buffer comprises Mg2+, PCR enhancer and stabilizer; the application adopts a mutant antibody to modify Taq DNA Polymerase, so that the enzyme activity can be released in a very short time under the condition of 95 DEG C; in addition, a shorter amplicon can greatly shorten the detection time, and the detection can be completed in about 55 min.

Description

Technical Field

[0001] This invention relates to the field of technology, and in particular to a lyophilized PCR reagent and kit for detecting nucleic acids in invasive pulmonary fungi. Background Technology

[0002] Invasive pulmonary fungal infections (IPFI) refer to infections caused by fungi invading the trachea, bronchi, and lung tissue, excluding lung lesions caused by fungal parasitism and allergies. In recent years, due to the widespread use of hematopoietic stem cell transplantation (HSCT), solid organ transplantation, the application of high-intensity immunosuppressants and high-dose chemotherapy drugs, and the internal intervention and placement of various catheters, the clinical incidence of IPFI has increased significantly. IPFI is also increasingly becoming one of the causes of death among organ transplant recipients, patients with malignant hematological diseases and malignant tumors, and other critically ill patients.

[0003] The most common invasive pulmonary fungi causing IPFI are Aspergillus, Cryptococcus, and Pneumocystis. Diseases caused by these fungi have insidious onset and atypical clinical symptoms, making early diagnosis difficult. Traditional laboratory diagnostic methods are too time-consuming, potentially missing the optimal treatment window, and also have low sensitivity. Therefore, addressing this problem technically is crucial.

[0004] 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.

[0005] 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 quantitative PCR (qPCR) targets specific conserved sequences in the fungal genome for in vitro qualitative detection of fungal nucleic acids in human lung samples. This method can accurately, sensitively, and rapidly detect fungal infections. It is simple to operate, requiring only one step to obtain results, and is characterized by its convenience, speed, and accuracy. It can detect patients in the window period or with low fungal loads, enabling early identification of infected individuals. It is relatively suitable for widespread clinical application and is of great significance for assisting in diagnosis and conducting large-scale population screening.

[0006] Most commercially available multiplex quantitative PCR kits for detecting invasive pulmonary fungal nucleic acids are liquid reagents containing temperature-sensitive Taq enzymes, primers, and probes. They require storage at -15°C or below to maintain their detection activity. Increased temperatures and repeated freeze-thaw cycles during storage, transportation, and use will reduce the reagents' detection performance. Furthermore, they are cumbersome to prepare, complex to operate, and have high storage and transportation costs. Additionally, the long detection time hinders rapid results and delays diagnosis and treatment. Summary of the Invention

[0007] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a freeze-dried PCR reagent and kit for detecting invasive pulmonary fungi.

[0008] The present invention also provides a lyophilized PCR reagent for detecting the above-mentioned invasive pulmonary fungi, comprising: ACP reaction lyophilized powder, ACP positive control and ACP negative control;

[0009] The ACP reaction lyophilized powder includes PCR Buffer, dNTP Mix, Taq DNA Polymerase, lyophilization protectant, and specific primers and probes;

[0010] The PCR Buffer contains Mg2+, PCR enhancer, and stabilizer.

[0011] The lyophilized PCR reagent for detecting invasive lung fungi according to the present invention includes the following specific primers and probes: oligonucleotide upstream and downstream primers and oligonucleotide fluorescent probes for specifically amplifying the conserved region of the 18S rRNA gene in the Aspergillus genome; oligonucleotide upstream and downstream primers and oligonucleotide fluorescent probes for specifically amplifying the conserved region of the ITS gene in the Cryptococcus neoformans genome; oligonucleotide upstream and downstream primers and oligonucleotide fluorescent probes for specifically amplifying the conserved region of the mtLSU rRNA gene in the Pneumocystis jirovecii genome; and oligonucleotide upstream and downstream primers and oligonucleotide fluorescent probes for specifically amplifying the human β-actin gene.

[0012] According to the lyophilized PCR reagent for detecting invasive pulmonary fungi nucleic acid, the oligonucleotide fluorescent probe is labeled with a fluorescent group and a quencher group at both ends, respectively. The fluorescent group is one of FAM, VIC, CY5, and ROX, and the quencher group is one of BHQ1, BHQ2, BHQ3, TAMRA, and MGB.

[0013] According to the lyophilized PCR reagent for detecting invasive lung fungi nucleic acid according to the present invention, each tube of ACP reaction lyophilized powder comprises: 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, and lyophilization protectant at a m / v of 1%-10%, 0.1-1 mM dNTP Mix, and 1-10× PCR Buffer.

[0014] According to the lyophilized PCR reagent for detecting invasive lung fungi nucleic acid according to the present invention, each tube of ACP reaction lyophilized powder comprises: 0.3 μM oligonucleotide upstream primer, 0.3 μM oligonucleotide downstream primer, 0.15 μM oligonucleotide fluorescent probe, 0.6 units of Taq DNA Polymerase, lyophilization protectant with a m / v of 2.5%, 0.6 mM dNTP Mix, and 5×PCRBuffe.

[0015] According to the lyophilized PCR reagent for detecting invasive pulmonary fungi nucleic acid according to the present invention, the Taq DNA Polymerase is a mutant Taq DNA Polymerase, and the enzyme is one of chemical modification, antibody modification, or ligand modification.

[0016] The lyophilized PCR reagent for detecting invasive pulmonary fungi nucleic acid according to the present invention uses pullulan, glycine, HPMC-E5, mannitol, trehalose, PEG-20000, sucrose, maltodextrin, gelatin, CMC-Na (50-200cps), PVP-10000, and other one or more of these as a lyophilization protectant.

[0017] According to the lyophilized PCR reagent for detecting invasive pulmonary fungi, the ACP positive control comprises a plasmid containing the 18S rRNA target gene fragment of Aspergillus fumigatus, a plasmid containing the ITS target gene fragment of Cryptococcus neoformans, a plasmid containing the mtLSU rRNA target gene fragment of Pneumocystis jirovecii, and a plasmid containing the human β-actin gene fragment, wherein the concentration of each target gene plasmid is 10. 5 The human β-actin gene fragment plasmid concentration was 10 copies / mL. 6 copies / mL.

[0018] According to the lyophilized PCR reagent for detecting invasive pulmonary fungi, the ACP negative control is a plasmid containing a human β-actin gene fragment, wherein the concentration of the human β-actin gene fragment plasmid is 10. 6 copies / mL.

[0019] The kit for detecting lyophilized PCR reagents for invasive pulmonary fungi according to the present invention comprises: the reagent composition of claim 1.

[0020] Beneficial effects:

[0021] (1) The present invention uses a mutant antibody to modify Taq DNA Polymerase to release enzyme activity in a very short time at 95°C. Combined with a shorter amplicon, the detection time can be greatly shortened, and the detection can be completed in about 55 minutes.

[0022] (2) The ACP reaction lyophilized powder prepared in this invention comprises oligonucleotide primers and probes, Taq DNA Polymerase, dNTPs, PCR Buffer, lyophilization protectant, etc., which are stable and coexist at room temperature. Its detection performance is consistent with that of the liquid reagent, and it can be transported at room temperature. This significantly reduces the transportation and storage costs of fluorescent PCR reagents, extends shelf life, reduces operational steps, simplifies the clinical testing process, and makes it more convenient for practical use and promotion.

[0023] (3) This invention designs specific primers and probes targeting the conserved regions of the Aspergillus spp. 18S rRNA gene, Cryptococcus neoformans ITS gene, and Yersinia natans mtLSU rRNA gene, enabling rapid in vitro detection of these three invasive lung fungi in vitro. The detection sensitivity reaches 200 copies / mL with high specificity. Simultaneously, the conserved region sequence of the human β-actin gene is used as an internal standard, allowing for full monitoring of the entire detection process, including sampling, nucleic acid extraction, and amplification, avoiding false negative results due to sampling, extraction, or amplification failures. Attached Figure Description

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0025] Figure 1 This is a structural diagram of the reaction lyophilized powder used in the lyophilized PCR reagent and kit for detecting invasive pulmonary fungi of the present invention.

[0026] Figure 2 This is a comparison chart of the detection performance of the ACP reaction lyophilized powder and liquid PCR reagent of the lyophilized PCR reagent and kit for detecting invasive pulmonary fungi in this invention.

[0027] Figure 3 The figure shows the stability test results of the lyophilized PCR reagent and kit for detecting invasive pulmonary fungi of the present invention.

[0028] Figure 4 This is an example of clinical sample detection results for the lyophilized PCR reagent and kit for detecting invasive pulmonary fungi according to the present invention. Detailed Implementation

[0029] 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.

[0030] Example 1

[0031] Primer and probe design for targets and internal standards

[0032] The 18S rRNA gene sequences of Aspergillus species (Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidus, Aspergillus versicolor, Aspergillus oryzae, Aspergillus glaucus, Aspergillus polyploid, and Aspergillus glemianum) and Cryptococcus neoformans (Cryptococcus neoformans, Cryptococcus grube, Cryptococcus neoformans Grubby variant, and Cryptococcus neoformans-Grube hybrid) were searched on NCBI (National Center of Biotechnology Information) over the past 10 years. The mtLSU rRNA gene sequence of Pneumocystis jirovecii and the human β-actin gene sequence were also searched. 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, and then the sequences were diluted to the required concentration as needed. The primer and probe sequences for the target and internal standard used in this invention are shown in the table below.

[0033]

[0034] Example 2

[0035] Preparation of lyophilized PCR reagents and kits for nucleic acid detection of invasive pulmonary fungi

[0036] (1) ACP reaction lyophilized powder

[0037] 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.

[0038] The ACP reaction lyophilized powder is prepared as follows:

[0039] Components Dosage per person (μL) Final concentration ASP-F (100μM) 0.075 0.3μM ASP-R (100μM) 0.075 0.3μM ASP-P (100μM) 0.0375 0.15μM Cry-F (100μM) 0.075 0.3μM Cry-R (100μM) 0.075 0.3μM Cry-P (100μM) 0.0375 0.15μM PJ-F (100μM) 0.075 0.3μM PJ-R (100μM) 0.075 0.3μM PJ-P (100μM) 0.0375 0.15μM Actin-F (100μM) 0.075 0.3μM Actin-R (100μM) 0.075 0.3μM Actin-P (100μM) 0.0375 0.15μM Lyophilization protectant stock solution (25%) 2.5 2.5％ TaqDNA Polymerase (5 units) 3 0.6 units dNTPMix(10mM) 1.5 0.6mM 5×PCRBuffer 5 1× <![CDATA[ddH2O]]> 12.25 / Total 25

[0040] After thoroughly mixing the above components, a final reaction volume of 25 μL of ACP reaction lyophilized powder solution 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 ACP 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 ACP reaction lyophilized powder.

[0041] (2) ACP positive controls: plasmids containing the 18S rRNA target gene fragment of Aspergillus fumigatus, plasmids containing the ITS target gene fragment of Cryptococcus neoformans, plasmids containing the mtLSU rRNA target gene fragment of Pneumocystis jirovecii, and plasmids containing the human β-actin gene fragment. The concentration of the target gene plasmids was 10. 5 copies / mL; human β-actin gene fragment plasmid concentration was 10. 6 Copies / mL, Volume: 300 μL / tube.

[0042] (3) ACP negative control: plasmid containing the human β-actin gene fragment, with a plasmid concentration of 10. 6 Copies / mL, Volume: 300 μL / tube.

[0043] The kit (48 doses / box) components are shown in the table below:

[0044]

[0045] Example 3

[0046] Detection Procedure for Lyophilized PCR Reagents and Kits for Nucleic Acid Detection of Invasive Pulmonary Fungi

[0047] 1. Sample pretreatment

[0048] 1.1 Sputum

[0049] 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.

[0050] 1.2 Bronchoalveolar lavage fluid

[0051] Clarified sample: No liquefaction is required; it can be used directly as the sample to be tested.

[0052] 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.

[0053] 2. Sample processing

[0054] Take 400 μL of the sample to be tested, an ACP positive control, and an ACP 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.

[0055] 3. Sample addition

[0056] 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 ACP reaction lyophilized powder, centrifuge for 30 seconds to ensure that the ACP reaction lyophilized powder is discharged to the bottom of the tube, and set aside for later use.

[0057] 3.2 Sample addition: Gently open the cap of the eight-tube containing the lyophilized powder, transfer 25 μL of the extracted test sample, ACP positive control and ACP negative control nucleic acid into the eight-tube, and tighten the cap.

[0058] 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.

[0059] 4. PCR amplification: This invention uses an ABI 7500 real-time quantitative PCR instrument for detection. Pre-denaturation is performed at 95℃ for 30s, followed by 40 cycles of amplification (denaturation at 95℃ for 10s, annealing and extension at 58℃ for 30s fluorescence collection). Aspergillus species are detected in the FAM channel, Cryptococcus neoformans in the ROX channel, Pneumocystis jirovecii in the CY5 channel, and human β-actin in the VIC channel.

[0060] 5 Results Analysis

[0061] 5.1 Quality Control Standards

[0062] ACP positive control: The Ct value of the target corresponding to the FAM, ROX, VIC and CY5 channels should be ≤35;

[0063] ACP negative control: No amplification curve (No Ct) or Ct value > 38 for FAM, ROX and CY5 channels, and Ct value of the corresponding target for VIC channel should be ≤ 35;

[0064] 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.

[0065] 5.2 Result Determination

[0066]

[0067] For samples that test positive for Aspergillus, Cryptococcus neoformans, or Pneumocystis jirovecii, 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.

[0068] Example 4

[0069] Comparison of detection performance between ACP lyophilized powder and liquid PCR reagent

[0070] The ACP reaction lyophilized powder used in this embodiment is the ACP reaction lyophilized powder for nucleic acid detection of invasive lung fungi prepared in Example 2, and the control reagent is liquid PCR reagent (with the same formulation as the ACP reaction lyophilized powder).

[0071] ACP Reaction Lyophilized Powder: ACP reaction lyophilized powder + 10 μL sample to be tested + 15 μL ddH2O Liquid PCR Reagent Amplification System: Prepare the liquid amplification system according to the table below, vortex to mix, and dispense 15 μL / well.

[0072]

[0073]

[0074] The templates were the ACP positive control, ACP negative control, and sputum samples from normal individuals prepared in Example 2.

[0075] The detection method was the same as in Example 3. The results of the ACP positive control and ACP negative control met the quality control standards, and the test was valid.

[0076] ACP reaction 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 nucleic acids of invasive lung fungi has the same detection performance as the liquid PCR reagent (with the same formulation as the ACP reaction lyophilized powder).

[0077] Example 5

[0078] Detection limit test of lyophilized PCR reagents and kits for nucleic acid detection of invasive pulmonary fungi.

[0079] The templates consisted of five different concentrations of Aspergillus fumigatus / Cryptococcus glottii culture strains and Pneumocystis jirovecii plasmids. 25 μL of RNase-Free Water was used as a template-free control (NTC). All templates were replicated 20 times. The detection method was the same as in Example 3.

[0080] The results are shown in the table below: the detection sensitivity for Aspergillus spp. / Cryptococcus neoformans / Pneumocystis jirovecii was 200 copies / mL.

[0081] Results of the detection limit test for invasive pulmonary fungi

[0082]

[0083] Example 6

[0084] Stability testing of lyophilized PCR reagents and kits for nucleic acid detection of invasive pulmonary fungi.

[0085] The kit used in this embodiment is the lyophilized PCR reagent and kit for detecting invasive pulmonary fungi 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 (preparation method same as in Example 2). The samples to be tested included one Aspergillus sputum sample positive, one Cryptococcus neoformans sample positive, one Pneumocystis jirovecii sample positive, and three negative sputum samples remaining from the hospital's laboratory. The detection method was the same as in Example 3.

[0086] Test results are as follows Figure 3 As shown, the amplification performance of the lyophilized PCR reagents and kits stored at room temperature for 12 months was consistent with that of the newly prepared lyophilized PCR reagents and kits in the control group. Therefore, the lyophilized PCR reagents and kits for the detection of nucleic acids from invasive lung fungi prepared in Example 2 can be stored at room temperature for 12 months.

[0087] Example 7

[0088] Application of lyophilized PCR reagents and kits for nucleic acid detection of invasive pulmonary fungi in clinical samples

[0089] The reagents used were the lyophilized PCR reagents and kits for detecting invasive pulmonary fungi prepared in Example 2. The samples to be tested were 2 Aspergillus positive sputum samples, 2 Cryptococcus neoformans positive sputum samples, 2 Pneumocystis jirovecii positive sputum samples, and 5 negative sputum samples remaining from the hospital's laboratory.

[0090] The detection method was the same as in Example 3. The results of the ACP positive control and ACP negative control met the quality control standards, and the test was valid. The test results are shown in the table below: 2 samples were positive for Aspergillus spp. ( Figure 4 Example); 2 samples were positive for Cryptococcus neoformans ( Figure 4 Example); 2 samples were positive for Pneumocystis jirovecii ( Figure 4 Example); 5 samples were negative ( Figure 4 Example); consistent with the hospital's diagnosis.

[0091]

[0092] 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 fungi, characterized in that, include: ACP reaction lyophilized powder, ACP positive control and ACP negative control; The ACP reaction lyophilized powder includes PCR Buffer, dNTP Mix, Taq DNA Polymerase, lyophilization protectant, and specific primers and probes; The PCR Buffer contains Mg2+, PCR enhancer, and stabilizer.

2. The lyophilized PCR reagent for detecting invasive pulmonary fungi according to claim 1, characterized in that, The specific primers and probes include: oligonucleotide upstream and downstream primers and oligonucleotide fluorescent probes for specifically amplifying the conserved region of the 18S rRNA gene in the Aspergillus genome; oligonucleotide upstream and downstream primers and oligonucleotide fluorescent probes for specifically amplifying the conserved region of the ITS gene in the Cryptococcus neoformans genome; oligonucleotide upstream and downstream primers and oligonucleotide fluorescent probes for specifically amplifying the conserved region of the mtLSU rRNA gene in the Pneumocystis jirovecii genome; and oligonucleotide upstream and downstream primers and oligonucleotide fluorescent probes for specifically amplifying the human β-actin gene.

3. The lyophilized PCR reagent for detecting invasive pulmonary fungi according to claim 2, 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.

4. The lyophilized PCR reagent for detecting invasive pulmonary fungi according to claim 1, characterized in that, Each tube of ACP 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 Mix, and 1-10× PCR Buffer.

5. The lyophilized PCR reagent for detecting invasive pulmonary fungi according to claim 1, characterized in that, Each tube of ACP reaction lyophilized powder contains: 0.3 μM oligonucleotide upstream primer, 0.3 μM oligonucleotide downstream primer, 0.15 μM oligonucleotide fluorescent probe, 0.6 units of Taq DNA Polymerase, lyophilization protectant at 2.5% m / v, 0.6 mM dNTPMix, and 5×PCR Buffe.

6. The lyophilized PCR reagent for detecting invasive pulmonary fungi according to claim 5, characterized in that, The Taq DNA Polymerase is a mutant Taq DNA Polymerase, and the enzyme is modified by one of the following methods: chemical modification, antibody modification, or ligand modification.

7. The lyophilized PCR reagent for detecting invasive pulmonary fungi according to claim 1, characterized in that, The freeze-drying protectant is one or more of pullulan, glycine, HPMC-E5, mannitol, trehalose, PEG-20000, sucrose, maltodextrin, gelatin, CMC-Na (50-200cps), PVP-10000, etc.

8. The lyophilized PCR reagent for detecting invasive pulmonary fungi according to claim 1, characterized in that, The ACP positive controls consisted of plasmids containing the 18S rRNA target gene fragment of Aspergillus fumigatus, plasmids containing the ITS target gene fragment of Cryptococcus neoformans, plasmids containing the mtLSU rRNA target gene fragment of Pneumocystis jirovecii, and plasmids containing the human β-actin gene fragment. The concentration of each target gene plasmid was 10⁻⁶. 5 The human β-actin gene fragment plasmid concentration is 10⁶ copies / mL.

9. The lyophilized PCR reagent for detecting invasive pulmonary fungi according to claim 1, characterized in that, The ACP negative control is a plasmid containing a human β-actin gene fragment, with a concentration of 10-1. 6 copies / mL.

10. A kit for detecting lyophilized PCR reagents for invasive pulmonary fungi, characterized in that, The kit comprises the reagent composition of claim 1.

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