Compositions, kits and uses thereof for detecting different types of fungi

Abstract

The present application belongs to the field of molecular biology detection, and particularly relates to a composition, method and use for simultaneous detection and differentiation of Pneumocystis jirovecii, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Lichtheimia ramosa, Cryptococcus neoformans, Candida albicans and Histoplasma capsulatum. The present application uses a multiplex PCR technique and a Qsep400 high-throughput analysis system, and the lengths of eight fungal amplification fragments are at least 20 bases apart, so that the target fragments can be accurately differentiated. Pneumocystis jirovecii, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Lichtheimia ramosa, Cryptococcus neoformans, Candida albicans and Histoplasma capsulatum can be simultaneously detected in one tube of reaction solution. One sample only needs to be subjected to nucleic acid extraction and simultaneously subjected to single-tube PCR amplification, and the sample detection result can be obtained through band analysis on the Qsep400, and the PCR process is not interfered, and the detection result is accurate.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically relating to compositions, methods, and uses for the joint detection and differentiation of Pneumocystis jirovecii, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Basilella marneffei, Cryptococcus neoformans, Candida albicans, and Histoplasma capsulatum. Background Technology

[0002] In recent years, with the widespread use of broad-spectrum antibiotics, glucocorticoids, and immunosuppressants, and the extensive implementation of organ transplantation and various invasive diagnostic and treatment procedures, special infections, especially deep fungal infections, have become increasingly common. The lungs, as the most susceptible site, are experiencing a continuously increasing prevalence. Simultaneously, the rate of pulmonary fungal infection in immunocompetent individuals is also showing a year-on-year increasing trend. Fungal infections often occur secondary to other serious primary diseases, and their clinical manifestations mostly present as symptoms of the primary disease. Clinical symptoms of pulmonary fungal infections include cough, fever, and dyspnea, which are nonspecific, making early diagnosis of pulmonary fungal infections quite difficult, with a high probability of missed or misdiagnosis. Common fungi causing pulmonary infections include Pneumocystis jirovecii, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Basilella marneffei, Cryptococcus neoformans, Candida albicans, and Histoplasma capsulatum. However, different pulmonary infections may differ in pathogenesis, clinical manifestations, severity, and prognosis. What they share is the difficulty in differential diagnosis, and they often cause relatively severe infections with poor prognosis and high mortality rates.

[0003] Currently, fungal culture is the "gold standard" for the clinical diagnosis of pulmonary fungal infections. However, its long culture period, low detection rate, and susceptibility to contamination can lead to a high false-positive rate, making it unsuitable as a commonly used clinical examination method. Typical CT imaging can serve as one of the auxiliary diagnostic criteria, but it needs to be combined with underlying diseases for preliminary diagnosis. Besides clinical manifestations, medical history, and imaging data, the diagnosis of pulmonary fungal infections mainly relies on laboratory pathogen detection. The most common molecular biological diagnostic method is qPCR, which shortens the time required from sampling to diagnosis. However, its application in fungal detection is relatively limited. One major reason is that fungal cell walls are thick, making nucleic acid extraction difficult under conventional methods, which greatly limits sensitivity and makes it difficult to achieve truly high-throughput detection. Another reason is that it also suffers from low throughput and a high false-positive rate.

[0004] Therefore, there is a need in this field for an early, rapid, and accurate etiological diagnosis, which is key to improving the prognosis of pulmonary fungal infections and reducing mortality. Summary of the Invention

[0005] In view of this, in a first aspect, the present invention provides a composition for detecting and distinguishing different types of fungi, comprising:

[0006] The upstream and downstream primers for detecting Bassula marneffei, as shown in SEQ ID NO:1-2;

[0007] As shown in SEQ ID NO:3-4, upstream and downstream primers for detecting Aspergillus flavus;

[0008] As shown in SEQ ID NO:5-6, the upstream and downstream primers for detecting Aspergillus niger;

[0009] For example, the upstream and downstream primers for detecting Aspergillus fumigatus shown in SEQ ID NO:7-8;

[0010] As shown in SEQ ID NO:9-10, upstream and downstream primers for detecting Cryptococcus neoformans;

[0011] As shown in SEQ ID NO:10-12, upstream and downstream primers for detecting Candida albicans;

[0012] As shown in SEQ ID NO:13-14, the upstream and downstream primers for detecting Histoplasma capsulatum; and

[0013] The upstream and downstream primers for detecting Pneumocystis jirovecii are shown in SEQ ID NO:15-16.

[0014] This invention employs multiplex PCR technology and the Qsep400 high-throughput analysis system. By designing primers for each pathogen and ensuring no interference between primers, and based on the Qsep400 electrophoresis principle, the lengths of eight fungal amplification fragments differ by at least 20 base pairs, achieving precise differentiation of the target fragments. It can simultaneously detect *Pneumocystis jirovecii*, *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus niger*, *Basilella marneffei*, *Cryptococcus neoformans*, *Candida albicans*, and *Histoplasma capsulatum* in a single reaction tube. For each sample, only one nucleic acid extraction and simultaneous single-tube PCR amplification are required. The sample detection results can be obtained through band analysis on the Qsep400, with no interference during the PCR process and accurate detection results.

[0015] Furthermore, the composition includes upstream and downstream primers for detecting an internal standard.

[0016] In some specific implementation schemes, the internal control is a human internal control gene. In one specific implementation scheme, the internal control is a human housekeeping gene.

[0017] Furthermore, in some embodiments, the compositions of the present invention may simultaneously include one or more of the primer pairs described above. In the present invention, a "pair" refers to mutually matching upstream and downstream primers that detect a target.

[0018] The compositions of this invention can be arbitrarily combined to detect any combination of the eight target sites. Those skilled in the art can combine them as needed, determining which target sites to detect by combining primer pairs corresponding to those target sites. All such combinations are included in this invention.

[0019] For example, it can include any 7 pairs of the above 8 primer pairs, any 6 pairs of the 8 primer pairs, any 5 pairs of the 8 primer pairs, any 4 pairs of the 8 primer pairs, any 3 pairs of the 8 primer pairs, any 8 pairs of the 8 primer pairs, or any 1 pair of the 8 primer pairs.

[0020] In some specific embodiments, the compositions of the present invention are used for PCR.

[0021] In one specific embodiment, each component of the composition of the present invention is contained in a separate package.

[0022] In one specific embodiment, the components of the composition of the present invention are contained in the same package.

[0023] Furthermore, the components of the composition of the present invention exist in a mixed form.

[0024] Secondly, the present invention provides the use of the above-described composition of the present invention in the preparation of a kit for the detection and differentiation of different types of fungi, wherein the fungi are Pneumocystis jirovecii, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Basilella marneffei, Cryptococcus neoformans, Candida albicans, and Histoplasma capsulatum.

[0025] Thirdly, the present invention provides a kit for detecting and distinguishing different types of fungi, the kit comprising the composition of the present invention as described above.

[0026] Furthermore, the kit also includes negative and positive controls.

[0027] In one specific implementation scheme, the negative control is at least one of DEPC H2O, physiological saline, and internal standard gene. The positive control is at least one of Pneumocystis jirovecii, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Basilella marneffei, Cryptococcus neoformans, Candida albicans, and Histoplasma capsulatum fragment plasmid or fragment DNA.

[0028] Furthermore, the kit also includes dNTPs, PCR buffer, and Mg. 2+ At least one of them.

[0029] Furthermore, the kit also includes at least one of the following: a nucleic acid release reagent, a nucleic acid extraction reagent, and a DNA polymerase.

[0030] Furthermore, the kit also includes nucleic acid release reagents, nucleic acid extraction reagents, dNTPs, DNA polymerase, PCR buffer, and Mg2+. 2+ At least one of them.

[0031] Furthermore, the concentration of the DNA polymerase is 3 U / reaction to 15 U / reaction, for example, the DNA polymerase can be Taq polymerase.

[0032] In one specific embodiment, the kit of the present invention includes: Taq enzyme, Mg 2+ dNTPs, primers, and PCR buffer.

[0033] Common PCR buffers consist of buffer systems such as Tris-HCl, MgCl2, KCl, and Triton X-100. The total volume in a single PCR reaction tube is typically 5-15 μl.

[0034] Fourthly, the invention provides the use of a reagent for preparing and distinguishing different types of fungi, the use comprising the following steps:

[0035] 1) Extract or release nucleic acid from the sample to be tested;

[0036] 2) Perform PCR amplification on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;

[0037] 3) Obtain and analyze the results.

[0038] In this invention, the sample used for detection can be serum, blood, etc., but is not limited to these.

[0039] Furthermore, the reaction conditions for the PCR are as follows:

[0040] Pre-denaturation: 95°C for 120 seconds, 1 cycle; denaturation: 95°C for 30 seconds; annealing: 56°C for 30 seconds; extension: 72°C for 1 minute, 45 cycles; final extension at 72°C for 5 minutes, 1 cycle.

[0041] Furthermore, the obtained and analyzed results include capillary electrophoresis.

[0042] In one specific implementation, a method is provided for detecting and differentiating different types of fungi for non-diagnostic purposes, the method comprising the following steps:

[0043] 1) Extract or release nucleic acid from the sample to be tested;

[0044] 2) Perform PCR amplification on the nucleic acid obtained in step 1) using the composition or kit of the present invention as described above;

[0045] 3) Obtain and analyze the results.

[0046] Furthermore, the reaction conditions for the PCR are as follows:

[0047] Pre-denaturation: 95°C for 120 seconds, 1 cycle; denaturation: 95°C for 30 seconds; annealing: 56°C for 30 seconds; extension: 72°C for 1 minute, 45 cycles; final extension at 72°C for 5 minutes, 1 cycle.

[0048] Furthermore, the obtained and analyzed results include capillary electrophoresis. Attached Figure Description

[0049] Figure 1 The image shows the detection results of the composition of the present invention for eight types of fungi;

[0050] Figure 2 This is a graph showing the sensitivity detection results of the composition of the present invention;

[0051] Figure 3 This is a graph showing the specificity detection results of the composition of the present invention;

[0052] Figure 4 The graph shows the detection results of the comparative composition of this invention in detecting eight fungi; Detailed Implementation

[0053] The present invention will be described in detail below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0054] Example 1: Primers used in this invention

[0055] The primers used in this invention are shown in Table 1.

[0056] Table 1

[0057]

[0058]

[0059] Example 2: Method for detecting and differentiating different types of fungi

[0060] Reagent preparation:

[0061] Based on the number of samples to be tested, positive controls, and negative controls, take the corresponding amount of PCR reaction solution in proportion, mix thoroughly, centrifuge at 2000 rpm for 10 seconds, and then set aside.

[0062] Sample processing and loading

[0063] Take 200 μL of the sample to be tested, negative control, and positive control into a 1.5 mL centrifuge tube, and perform nucleic acid extraction using the nucleic acid extraction or purification reagents of Sansure Biotech Inc. in accordance with their instructions.

[0064] Take 2 μL each of the extracted nucleic acid, negative control, and positive control and add them to the corresponding 0.2 mL PCR reaction tubes.

[0065] PCR amplification

[0066] Prepare the multiplex PCR reaction solution according to Table 2. Add 5 μl of the reaction solution to each 0.2 mL PCR reaction tube and perform amplification according to Table 3 below.

[0067] Table 2

[0068] Components Volume / concentration in each reaction PCR buffer* 1μL dNTPs (100mM) 0.2μL <![CDATA[1mol / L MgCl2]]> 0.8μL Primers (1 μM) 2μL PCR Enzyme 0.5μL <![CDATA[ddH2O]]> 3.5μL DNA 2μL Total 10μL

[0069] Table 3

[0070]

[0071]

[0072] PCR product purification

[0073] After centrifugation, the PCR product was transferred to a 1.5 ml EP tube. An appropriate amount of magnetic bead solution was added according to the fragment length. The mixture was shaken and allowed to stand for 3 min.

[0074] After centrifuging the magnetic bead product mixture, place it on a magnetic rack and wait for the magnetic beads to aggregate. Then, discard the supernatant.

[0075] Add 200 μL of 80% ethanol, rotate and wash on a magnetic rack for 2-3 weeks, discard the supernatant; repeat the washing operation twice.

[0076] After centrifugation, place the tube on a magnetic rack, remove the residual liquid, open the cap and let it stand for 5 minutes to allow the residual ethanol in the tube to evaporate completely.

[0077] Add 25-50 μL of ddH2O, shake and let stand for 3 min, centrifuge and place on a magnetic rack. Take the supernatant as the purified product and freeze at -20℃ for later use.

[0078] Qsep400 on the machine

[0079] After the concentration of the PCR purified product was measured by a Qubit fluorescence spectrophotometer (Thermo Fisher), it was diluted to 2-5 ng / μL with ddH2O. 2 μL of the diluted product was mixed with 13 μL of Qsep 1x Dilution buffer, and the sample was added to an appropriate number of wells on a 96-well capillary electrophoresis plate for capillary electrophoresis separation.

[0080] Interpretation of test results

[0081] Fragment size analysis was performed on the results using the default parameters of the Qsep400 fully automated nucleic acid and protein analysis system's software. The horizontal axis represents fragment size, and the vertical axis represents signal strength. The spectra obtained from the Qsep400 analyzer's software were compared with standard spectra to determine the type of pathogen.

[0082] Example 3: Detection results of test samples of the composition of the present invention

[0083] The primers shown in Example 1 were used to detect and differentiate eight fungal samples according to the method in Example 2. The detection results are as follows: Figure 1 As shown in the figure, the composition of the present invention can detect and distinguish eight fungi in one tube.

[0084] Example 4: Sensitivity of the composition of the present invention

[0085] Eight plasmids synthesized by fungi were diluted to 500 copies / ml and 400 copies / ml, amplified, and detected. Figure 2 As shown, a mixed sample of 500 copies / ml can amplify all bands, showing the corresponding products of eight fungi; while a mixed sample of 400 copies / ml can amplify some bands, so the detection limit is determined to be 500 copies / ml.

[0086] Example 5: Specificity of the composition of the present invention

[0087] After mixing the eight fungi in this kit with positive samples including Rhizopus, Mucor, Rhizopus rhizopus, Sedorsporium, Fusarium oxysporum, Fusarium moniliforme, Penicillium citrinum, Penicillium chrysogenum, Moraxella catarrhalis, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, Mycoplasma pneumoniae, Acinetobacter baumannii, Escherichia coli, Candida tropicalis, Candida krusei, and human genomic DNA, amplification and detection were performed. The results still only showed products corresponding to the eight fungi. (See attached results). Figure 3No corresponding products were amplified for other cross-reactive pathogens, indicating that this kit does not cross-react with positive samples such as Rhizopus, Mucor, Rhizopus rumenella, Sardorius, Fusarium oxysporum, Fusarium moniliforme, Penicillium citrinum, Penicillium chrysogenum, Moraxella catarrhalis, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, Mycoplasma pneumoniae, Acinetobacter baumannii, Escherichia coli, Candida tropicalis, Candida krusei, and human genomic DNA.

[0088] Comparative Example 1: Other primers designed in this invention that did not perform well.

[0089] Due to the complementary base pairing principle, primers can form dimers, but this probability is very low and can be eliminated at the initial design stage. However, when detecting multiple pathogens together, there are many primers, and primers are prone to dimerization. To ensure the conservation of the design (conservatism is crucial for the accuracy of detection) and to consider the mutual interference between different primers, careful primer design is required.

[0090] Therefore, the inventors also designed other primers (sequences not shown) to form different detection systems, which were also used for eight fungi. Specific detection results are as follows: Figure 4 As shown, the results indicate that this detection system only produced partial amplification bands and could not completely detect the eight fungi; therefore, the overall detection effect was poor.

Claims

1. A composition for detecting and distinguishing different types of fungi, characterized in that, The composition consists of the following primers: The upstream and downstream primers for detecting *Basilella marneffei* are shown in SEQ ID NO:1~2; For example, the upstream and downstream primers for detecting Aspergillus flavus shown in SEQ ID NO:3~4; For example, the upstream and downstream primers for detecting Aspergillus niger shown in SEQ ID NO:5~6; For example, the upstream and downstream primers for detecting Aspergillus fumigatus shown in SEQ ID NO:7~8; For example, the upstream and downstream primers for detecting Cryptococcus neoformans shown in SEQ ID NO:9~10; For example, the upstream and downstream primers for detecting Candida albicans shown in SEQ ID NO:10~12; The upstream and downstream primers for detecting Histoplasma capsulatum, as shown in SEQ ID NO:13~14; and Upstream and downstream primers for detecting Pneumocystis jirovecii, as shown in SEQ ID NO:15~16.

2. The composition according to claim 1, characterized in that, The composition also includes upstream and downstream primers for detecting an internal standard.

3. The composition according to claim 2, characterized in that, The internal standard is a human internal standard gene.

4. The composition according to claim 1, characterized in that, The components of the composition are contained in the same package.

5. The composition according to claim 1, characterized in that, The components of the composition exist in a mixed form.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of a kit for the detection and differentiation of different types of fungi, wherein, The fungi mentioned are Pneumocystis jirovecii, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Basilella marneffei, Cryptococcus neoformans, Candida albicans, and Histoplasma capsulatum.

7. A kit for detecting and differentiating different types of fungi, said kit comprising the composition of any one of claims 1 to 5.

8. The reagent kit according to claim 7, characterized in that, The kit also includes negative and positive controls.

9. The reagent kit according to claim 8, characterized in that, The kit also includes nucleic acid release reagent, nucleic acid extraction reagent, dNTPs, DNA polymerase, PCR buffer, and Mg. 2+ At least one of them.

10. A method for detecting and differentiating different types of fungi for non-diagnostic purposes, the method comprising the following steps: 1) Extract or release nucleic acid from the sample to be tested; 2) Perform PCR on the nucleic acid obtained in step 1) using the composition as described in any one of claims 1 to 5 or the kit as described in any one of claims 7 to 9; 3) Obtain and analyze the results.

Images