Nucleic acid composition for detecting fungi as well as kit and detection method thereof

Abstract

The invention discloses a nucleic acid composition for detecting fungi as well as a kit and a detection method thereof, and relates to the technical field of molecular diagnosis. The embodiment of the invention provides a group of nucleic acid compositions, the compositions comprise primer pairs 1-5, the compositions can realize synchronous detection of aspergillus, cryptococcus, pneumocystis yarrowii and mucor on the basis of a real-time fluorescent quantitative PCR technology, and the nucleic acid compositions have the remarkable advantages of short detection time, simplicity and convenience in operation, high sensitivity, strong specificity and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular diagnostics, in particular, to a nucleic acid composition for detecting fungi, a kit and a detection method thereof. BACKGROUND

[0002] Aspergillus is a kind of fungus commonly existing in the environment, and invasive aspergillosis includes chronic pulmonary aspergillosis and invasive pulmonary aspergillosis.

[0003] Pulmonary cryptococcosis is an invasive pulmonary fungal disease caused by Cryptococcus neoformans or Cryptococcus gattii. Its clinical manifestations and imaging signs are both lack of specificity, and it is easy to be misdiagnosed as lung cancer, tuberculosis, viral pneumonia and other fungal pneumonia. Once the treatment is delayed, the cryptococcus spreads to the brain, which can lead to cryptococcal meningitis, and the patient's mortality rate is extremely high.

[0004] Pneumocystis jirovecii is an opportunistic pathogen that can cause Pneumocystis jirovecii pneumonia. Solid organ, stem cell transplant recipients and patients with acquired immunodeficiency syndrome who have not received effective prevention are high-risk groups for infection.

[0005] Mucormycosis caused by Mucorales fungi is a rapidly progressive invasive fungal disease, and its incidence rate is only second to Candida and Aspergillus infection in fungal infections. Among the five main infection sites of mucormycosis, the lung is the most common, and the mortality rate is as high as 56%. Due to the characteristics of rapid progression, misdiagnosis and high mortality, early diagnosis and treatment of mucormycosis has always been a big problem plaguing the clinic.

[0006] Invasive fungal infections are increasingly serious, and the current diagnostic methods (including direct microscopy, direct microscopy, serological diagnostic methods and molecular biology diagnostic methods) all have certain shortcomings, such as: long time-consuming, easy to delay clinical diagnosis and treatment; low detection sensitivity and insufficient specificity; high false positive rate of antigen-antibody detection method, and specific antibody is difficult to prepare, etc.

[0007] In view of this, the present application is proposed. SUMMARY

[0008] The present application relates to the field of molecular diagnostics, in particular, to a nucleic acid composition for detecting fungi, a kit and a detection method thereof.

[0009] The present application is implemented in the following manner:

[0010] In a first aspect, the present application provides a nucleic acid composition, which comprises: a combination of at least one or more of primer pairs 1-5; wherein the detection region of the primer pairs 1-5 is selected from at least 80% of the full length of the sequence as described in SEQ ID NO: 55-59.

[0011] In a second aspect, the embodiments of the present application provide a kit containing the nucleic acid composition according to the preceding embodiments.

[0012] In a third aspect, the embodiments of the present application provide use of the nucleic acid composition according to the preceding embodiments in the preparation of a kit for detecting fungi, including any one or more of Aspergillus, Cryptococcus, Pneumocystis jirovecii and Mucorales.

[0013] In a fourth aspect, the embodiments of the present application provide a method for detecting fungi, comprising: performing PCR detection on a sample to be tested using the nucleic acid composition according to the preceding embodiments or the kit according to the preceding embodiments; the detection is not directly for the diagnosis or treatment of a disease; and the fungi are those described in the preceding embodiments.

[0014] The present application has the following advantages:

[0015] The embodiments of the present application provide a set of nucleic acid compositions, which are based on real-time fluorescent quantitative PCR technology and can realize simultaneous detection of Aspergillus, Cryptococcus, Pneumocystis jirovecii and Mucorales, and have the advantages of short detection time, simple operation, high sensitivity and strong specificity. DETAILED DESCRIPTION

[0016] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If specific conditions are not specified in the embodiments, conventional conditions or manufacturer recommended conditions are used. If the used reagents or instruments are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0017] The present application is directed to specific regions of Aspergillus, Cryptococcus, Pneumocystis jirovecii and Mucorales, and a primer probe combination is designed to realize simultaneous detection of four indicators in one sample, i.e., identification of Aspergillus, Cryptococcus, Pneumocystis jirovecii and Mucorales at the same time, and high detection limit in a multiple system. The primer probe, DNA polymerase, dNTPs, PCR buffer and the like are freeze-dried using a vacuum freeze-drying technology, and exist in the form of freeze-dried balls in the kit. Finally, the freeze-dried balls are reconstituted and amplified using the eluted product of the extracted nucleic acid, and closed amplification is performed on a microfluidic chip.

[0018] In one aspect, the embodiments of the present application provide a nucleic acid composition, comprising: a combination of at least one or more of primer pairs 1-5; wherein the detection region of the primer pairs 1-5 is selected from at least 80% of the full length of the sequence according to SEQ ID NO: 55-59.

[0019] In some embodiments, the at least 80% comprises 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or more, or 100%.

[0020] In some embodiments, the sequences of the upstream primer and the downstream primer of primer pair 1 are set forth in SEQ ID NOs: 1-2, 19-20, or 37-38, in order; the sequences of the upstream primer and the downstream primer of primer pair 2 are set forth in SEQ ID NOs: 4-5, 22-23, or 40-41, in order; the sequences of the upstream primer and the downstream primer of primer pair 3 are set forth in SEQ ID NOs: 7-8, 25-26, or 43-44, in order; the sequences of the upstream primer and the downstream primer of primer pair 4 are set forth in SEQ ID NOs: 10-11, 28-29, or 46-47, in order; the sequences of the upstream primer and the downstream primer of primer pair 5 are set forth in SEQ ID NOs: 13-14, 31-32, or 49-50.

[0021] In some embodiments, the sequences of the upstream primer and the downstream primer of primer pair 1-5 are set forth in SEQ ID NOs: 1-2, 4-5, 7-8, 10-11, and 13-14; or SEQ ID NOs: 19-20, 22-23, 25-26, 28-29, and 31-32; or SEQ ID NOs: 37-38, 40-41, 43-44, 46-47, and 49-50, in order.

[0022] In some embodiments, the nucleic acid composition further comprises a probe.

[0023] In some embodiments, the probe further comprises any one or more of probes 1-5. The sequence of the probe 1 is set forth in SEQ ID NO: 3, 21, or 39; the sequence of the probe 2 is set forth in SEQ ID NO: 6, 24, or 42; the sequence of the probe 3 is set forth in SEQ ID NO: 9, 27, or 45; the sequence of the probe 4 is set forth in SEQ ID NO: 12, 30, or 48; the sequence of the probe 5 is set forth in SEQ ID NO: 15, 33, or 51.

[0024] Primer pairs 1-2 and probes 1-2 are designed for the conserved sequences of mtDNA and 28s rRNA of Aspergillus genus, and can be used for universal detection of Aspergillus genus, not limited to the detection of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Aspergillus versicolor, Aspergillus candidus, Aspergillus sydowii, Aspergillus glaucus, Aspergillus oryzae, Aspergillus japonicus, Aspergillus ochraceus, and other Aspergillus species.

[0025] The primer pair 3 and the probe 3 are designed for the ITS conserved sequence of Cryptococcus, and can realize the detection of any one or more of Cryptococcus neoformans and Cryptococcus gattii.

[0026] The primer pair 4 and the probe 4 are designed for the specific fragment of mtLSU rRNA of Pneumocystis jirovecii.

[0027] The primer pair 5 and the probe 5 are designed for the conserved sequence of 18s rRNA of Mucorales, and can be used for the universal detection of Mucorales, and are not limited to the detection of various Mucorales species such as Rhizopus microsporus, Rhizopus arrhizus, Rhizopus delemar, Lichtheimia corymbifera, Lichtheimia profilica, Lichtheimia curvatus, Mucor indicus, Mucor circinelloides, Mucor pusillus, and Mucor miehei.

[0028] In some embodiments, the 5' end of the probe is connected with a fluorescent reporter group, and the 3' end is connected with a fluorescent quencher group and / or MGB.

[0029] In some embodiments, the fluorescent reporter group is selected from any one of FAM, Texas Red, Cy5, Cy5.5, CY7, or HEX.

[0030] In some embodiments, the fluorescent quencher group is selected from any one of BHQ2 and TAMRA.

[0031] In some embodiments, the nucleic acid composition further comprises a primer pair 6 and / or a probe 6 for detecting an internal reference.

[0032] In some embodiments, the internal reference is an exogenous bacterial species Bacillus subtilis, which is involved in the whole process of nucleic acid extraction and PCR amplification, so as to realize the monitoring of the whole process.

[0033] In some embodiments, the sequences of the upstream primer and the downstream primer of the primer pair 6 are shown in SEQ ID NO: 16-17, 34-35, or 52-53.

[0034] In some embodiments, the sequences of the upstream primer and the downstream primer of the probe 6 are shown in SEQ ID NO: 18, 36, or 54.

[0035] In another aspect, the embodiments of the present application also provide a kit containing the nucleic acid composition of any of the foregoing embodiments.

[0036] In some embodiments, the kit further comprises at least one of a PCR reaction solution, a positive quality control, and a negative quality control.

[0037] In some embodiments, the nucleic acid composition, the PCR reaction solution, the positive quality control, and the negative quality control are in the form of lyophilized products.

[0038] In some embodiments, the PCR reaction solution comprises at least one of Taq enzyme, dNTPs, and a buffer containing magnesium ions.

[0039] In another aspect, the embodiments of the present application also provide use of the nucleic acid composition according to any of the preceding embodiments in the preparation of a kit for detecting fungi, which include any one or more of Aspergillus, Cryptococcus, Pneumocystis jirovecii, and Mucorales.

[0040] In some embodiments, the Aspergillus includes any one or more of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Aspergillus versicolor, Aspergillus candidus, Aspergillus sydowii, Aspergillus glaucus, Aspergillus oryzae, and Aspergillus ochraceus.

[0041] The Cryptococcus includes Cryptococcus neoformans, which includes any one or more of var. gattii, var. neoformans, and var. mixed.

[0042] The Mucorales includes any one or more of Rhizopus microsporus, Rhizopus arrhizus, Rhizopus delemar, Lichtheimia corymbifera, Lichtheimia profilera, Mucor circinelloides, Mucor indicum, Mucor mucedo, Mortierella minutissima, and Lichtheimia exsuccosa.

[0043] In another aspect, the embodiments of the present application also provide a method for detecting fungi, which comprises performing PCR detection on a sample to be tested using the nucleic acid composition according to any of the preceding embodiments or the kit according to any of the preceding embodiments; the fungi are the fungi according to any of the preceding embodiments.

[0044] In some embodiments, the upstream primer and / or the downstream primer (i.e. each of the upstream primer and the downstream primer) in the nucleic acid composition has a working concentration of 0.01-10 μM in the amplification system, which can be specifically any one or a range between any two of 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μM.

[0045] In some embodiments, the probe in the nucleic acid composition has a working concentration of 0.01-10 μM in the amplification system, which can be specifically any one or a range between any two of 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μM.

[0046] In some embodiments, the molar ratio of the upstream primer to the downstream primer is (1-5):(1-5); the molar ratio can be specifically any one of 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:5, 3:1, 3:2, 3:4, 3:5, 4:1, 4:3, 4:5, 5:1, 5:2, 5:3, 5:4 or a range between any two of them.

[0047] In some embodiments, the sample to be detected can be a blood sample, a tissue sample, an environmental sample or an artificially prepared positive sample.

[0048] The detection is not directly aimed at the diagnosis or treatment of a disease, for example, when the sample is an environmental sample or an artificially prepared positive sample, the direct purpose of the detection is to detect whether there is a coronavirus pathogen therein, rather than the diagnosis and treatment of a disease.

[0049] In some embodiments, the reaction procedure of PCR is as follows: 94-96℃ pre-denaturation for 30 seconds-10 minutes, 1 cycle; 94-96℃ denaturation for 0-15 seconds, 58-62℃ annealing and extension for 0-60 seconds, denaturation-annealing and extension for 40-60 cycles, and collection of fluorescence.

[0050] In some embodiments, the amplification system of PCR is as follows: 10-50 μL of DNA template, 20-60 μL of PCR amplification reagent; the PCR amplification reagent is prepared as follows: 0.01-10 μM of each of PCR amplification buffer (PCR reaction solution), primer and probe, and 10-200 μM of dNTPs.

[0051] The features and performances of the present application are further described in detail below in combination with embodiments.

[0052] Embodiment 1

[0053] This embodiment provides a primer-probe combination for detecting Aspergillus, Cryptococcus, Pneumocystis jirovecii and Mucorales by multiplex PCR, as shown in Table 1.

[0054] Table 1 First group of primer-probe combinations

[0055]

[0056] The above primer-probe combination is prepared into a PCR amplification reaction solution together with a PCR reaction buffer, dNTPs, etc. The specific preparation is as follows:

[0057] Reagent name Original concentration Final concentration Volume added 10X PCR amplification buffer 10× 1× 4.58 μL dNTPs 10 mM 84 μM 0.42 μL Upstream primer 100 μM 400 nM 0.2 μL each Downstream primer 100 μM 400 nM 0.2 μL each Probe 100 μM 200 nM 0.1 μL each Enzyme strip / / 32 μL PCR reaction volume / / 40 μL Sample volume / / 10 μL

[0058] PCR reaction procedure: 95℃, 2 minutes, 1 cycle; 95℃, 5 seconds, 60℃, 30 seconds, 40 cycles, and collection of fluorescence.

[0059] The Aspergillus fumigatus, Cryptococcus gattii, Pneumocystis jirovecii, and Lichtheimia corymbifera bacterial solutions were diluted to 2000, 1000, 500, 200, 100, and 50 copies / mL, respectively, and each concentration was detected 20 times, the Ct values were counted, the detection rate was calculated, and when the detection rate was ≥95%, the corresponding concentration was the detection limit concentration. The detection results are as follows.

[0060] Table 2 Aspergillus fumigatus detection results

[0061]

[0062]

[0063] Table 3 Cryptococcus gattii detection results

[0064]

[0065]

[0066] Table 4 Pneumocystis jirovecii detection results

[0067]

[0068] Table 5 Lichtheimia corymbifera detection results

[0069]

[0070]

[0071] Example 2

[0072] This example provides a primer probe combination for detecting Aspergillus, Cryptococcus, Pneumocystis jirovecii, and Mucorales by multiplex PCR, as shown in Table 6.

[0073] Table 6 Second group of primer probe combinations

[0074]

[0075] The PCR reaction system and reaction procedure are as described in Example 1.

[0076] The Aspergillus fumigatus, Cryptococcus gattii, Pneumocystis jirovecii, and Lichtheimia corymbifera bacterial solutions were diluted to 2000, 1000, 500, 200, 100, and 50 copies / mL, respectively, and each concentration was detected 20 times, the Ct values were counted, the detection rate was calculated, and when the detection rate was ≥95%, the corresponding concentration was the detection limit concentration. The detection results are as follows.

[0077] Table 7 Aspergillus fumigatus detection results

[0078]

[0079]

[0080] Table 8 Detection results of Cryptococcus gattii

[0081]

[0082] Table 9 Detection results of Pneumocystis jirovecii

[0083]

[0084]

[0085] Table 10 Detection results of Lichtheimia curreyae

[0086]

[0087]

[0088] Example 3

[0089] This example provides a primer probe combination for detecting Aspergillus, Cryptococcus, Pneumocystis jirovecii and Lichtheimia curreyae by multiplex PCR, as shown in Table 11.

[0090] Table 11 Third group of primer probe combinations

[0091]

[0092] The PCR reaction system and reaction procedure are the same as described in Example 1.

[0093] The Aspergillus fumigatus, Cryptococcus gattii, Pneumocystis jirovecii and Lichtheimia curreyae were diluted to 2000, 1000, 500, 200, 100, 50 copies / mL respectively, and each concentration was detected 20 times, the Ct value was counted, and the detection rate was calculated. When the detection rate is ≥95%, the corresponding concentration is the detection limit concentration. The detection results are as follows.

[0094] Table 12 Detection results of Aspergillus fumigatus

[0095]

[0096]

[0097] Table 13 Detection results of Cryptococcus gattii

[0098]

[0099] Table 14 Detection results of Pneumocystis jirovecii

[0100]

[0101]

[0102] Table 15 Detection results of Cladosporium cladosporioides

[0103]

[0104] Specificity of Example 4

[0105] The cross-reactive bacteria were diluted to 10 6 copies / mL, and PCR reaction solution was prepared and amplified according to the primer probe combinations shown in Examples 1-3 and the PCR reaction solution preparation, and the specific cross-reactive bacteria and detection results are as follows:

[0106] Table 16 Detection results of the first group of primer probes

[0107]

[0108] Table 17 Detection results of the second group of primer probes

[0109]

[0110]

[0111] Table 18 Detection results of the third group of primer probes

[0112]

[0113]

[0114] The above results show that the cross-reactive bacteria listed in Example 4 have no cross-reaction with the detection targets of the kit, and the specificity is good.

[0115] Example 5

[0116] 50 real clinical positive sputum samples were selected, and the primer probe combinations in Examples 1-3 above were used to prepare PCR reaction solution, and 50 samples were detected respectively, and the PCR reaction system and reaction program were the same as shown in Example 1. The detection results are as follows.

[0117] Table 19 Detection results

[0118]

[0119]

[0120]

[0121] As can be seen from the results, the first group to the third group of primer probes have good specificity.

[0122] The specific region of the first to third sets of primer probes is selected from the following.

[0123] Table 20 Specific region

[0124]

[0125] The above descriptions are only the preferred embodiments of the present application and are not intended to limit the present application. The present application can be variously changed and modified by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the scope of the present application.

Claims

1. A nucleic acid composition, characterized in that, It comprises; a combination of at least one or more of primer pairs 1-5; wherein the detection region of the primer pairs 1-5 is selected from at least 80% of the full length of the sequences as set forth in SEQ ID NOs: 55-59, respectively.

2. The nucleic acid composition of claim 1, wherein, The sequences of the upstream primer and the downstream primer of primer pair 1 are as set forth in SEQ ID NOs: 1-2, 19-20, or 37-38, respectively; The sequences of the upstream primer and the downstream primer of primer pair 2 are as set forth in SEQ ID NOs: 4-5, 22-23, or 40-41, respectively; The sequences of the upstream primer and the downstream primer of primer pair 3 are as set forth in SEQ ID NOs: 7-8, 25-26, or 43-44, respectively; The sequences of the upstream primer and the downstream primer of primer pair 4 are as set forth in SEQ ID NOs: 10-11, 28-29, or 46-47, respectively; The sequences of the upstream primer and the downstream primer of primer pair 5 are as set forth in SEQ ID NOs: 13-14, 31-32, or 49-50, respectively; Optionally, the sequences of the upstream primer and the downstream primer of the primer pairs 1-5 are as set forth in SEQ ID NOs: 1-2, 4-5, 7-8, 10-11, and 13-14, respectively; or SEQ ID NOs: 19-20, 22-23, 25-26, 28-29, and 31-32, respectively; or SEQ ID NOs: 37-38, 40-41, 43-44, 46-47, and 49-50, respectively.

3. The nucleic acid composition of claim 1 or 2, wherein The nucleic acid composition further comprises a probe; Optionally, the probe further comprises any one or more of probes 1-5; The sequence of the probe 1 is as set forth in SEQ ID NO: 3, 21, or 39; The sequence of the probe 2 is as set forth in SEQ ID NO: 6, 24, or 42; The sequence of the probe 3 is as set forth in SEQ ID NO: 9, 27, or 45; The sequence of the probe 4 is as set forth in SEQ ID NO: 12, 30, or 48; The sequence of the probe 5 is as set forth in SEQ ID NO: 15, 33, or 51; Optionally, the 5' end of the probe is linked with a fluorescent reporter group, and the 3' end is linked with a fluorescent quencher group and / or MGB; Optionally, the fluorescent reporter group is selected from any one of FAM, Texas Red, Cy5, Cy5.5, CY7, and HEX; Optionally, the fluorescent quencher group is selected from any one of BHQ2, TAMRA.

4. The nucleic acid composition according to any one of claims 1 to 3, wherein The nucleic acid composition further comprises primer pair 6 and / or probe 6 for detecting an internal reference; Optionally, the sequences of the upstream primer and the downstream primer of the primer pair 6 are as set forth in SEQ ID NOs: 16-17, 34-35, or 52-53; Optionally, the sequences of the upstream primer and the downstream primer of the probe 6 are as set forth in SEQ ID NO: 18, 36, or 54.

5. A kit characterized in that, It contains the nucleic acid composition of any one of claims 1-4.

6. The kit of claim 5, wherein The kit further comprises at least one of a PCR reaction solution, a positive quality control, and a negative quality control; Optionally, the PCR reaction solution comprises at least one of Taq enzyme, dNTPs, and a buffer containing magnesium ions.

7. Use of a nucleic acid composition according to any one of claims 1 to 4 for the preparation of a kit for the detection of fungi, including: Any one or more of Aspergillus, Cryptococcus, Pneumocystis jirovecii, and Mucorales.

8. Use according to claim 7, characterized in that, The Aspergillus includes any one or more of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Aspergillus versicolor, Aspergillus candidus, Aspergillus sydowii, Aspergillus glaucus, Aspergillus oryzae, and Aspergillus ochraceus; Optionally, the Cryptococcus includes Cryptococcus neoformans and / or Cryptococcus gattii; Optionally, the Cryptococcus neoformans includes any one or more of var. grubii, var. neoformans, and var. gattii. Optionally, the Mucorales includes any one or more of Rhizopus microsporus, Rhizopus arrhizus, Rhizopus delemar, Absidia corymbifera, Lichtheimia ramosa, Lichtheimia repens, Mucor circinelloides, Mucor indicus, Mucor mucedo, Mucor pusillus, and Lichtheimia exsuccosa.

9. A method for detecting fungi, characterized by, It includes: using the nucleic acid composition of any one of claims 1-4 or the kit of claim 5 or 6 to perform PCR detection on a sample to be tested; the detection is not directly for the diagnosis or treatment of a disease; and the fungus is the fungus described in claim 7 or 8.

10. The detection method according to claim 9, characterized in that, The upstream primer and / or the downstream primer in the nucleic acid composition has an effective concentration of 0.01-10 μM in the amplification system. Optionally, the probe in the nucleic acid composition has an effective concentration of 0.01-10 μM in the amplification system.

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