CN112626263B - Kit for detecting respiratory tract infection fungal pathogens at constant temperature by using enzyme digestion probe - Google Patents
Kit for detecting respiratory tract infection fungal pathogens at constant temperature by using enzyme digestion probe Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention provides a kit for detecting respiratory tract infection fungal pathogens by using an enzyme digestion probe at a constant temperature, wherein 8 pathogen targets are synthesized into three multiple nucleic acid reaction solutions by using respective primer probe sets. The nucleic acid detection kit provided by the invention can realize the multiplex isothermal amplification detection of 8 fungal pathogens with high frequency in respiratory tract infection by three single tubes, is simple and convenient to operate, can realize bedside detection, can realize the screening of various fungal pathogens in a sample to be detected within 30min during the whole detection process, and can realize the rapid and accurate screening of various fungal pathogens in the sample to be detected.
Description
Technical Field
The invention relates to the technical field of nucleic acid constant temperature detection, in particular to a kit for detecting respiratory tract infection fungal pathogens by using an enzyme digestion probe at a constant temperature.
Background
Respiratory tract infection belongs to common high-incidence diseases, and fungal infection in respiratory tract infection is mainly Candida, Aspergillus, Mucor, Yersinia pneumocystis and other species, wherein 5 species of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis and Candida krusei in Candida are 95% of fungal pathogen infection of human. In recent years, the use of broad-spectrum antibiotics causes the morbidity of fungal infection to rise year by year, and the mortality rate is high, so that the early and rapid specific diagnosis of fungal pathogens is realized, and the method is very necessary for improving the prognosis and survival rate of patients.
At present, microscopic examination culture is still used as a gold standard for diagnosing fungal infection clinically, but the culture needs a long period, microscopic examination or pathological examination has high requirements on inspection personnel, and high false negative and false positive exist, so that misdiagnosis and missed diagnosis are easily caused, the illness state of a patient is delayed, and certain limitations are realized in clinical application. The nucleic acid detection pathogen is more efficient, rapid and strong in specificity, and with the continuous development of molecular biology theory and technology, the nucleic acid isothermal amplification technology can complete the amplification of DNA or RNA at a constant temperature.
However, most of the existing nucleic acid isothermal amplification methods can only perform single-tube single-multiplex detection, which is time-consuming, labor-consuming and high in detection cost when performing screening detection of multiple targets. Therefore, the establishment of a reagent for performing multiple detection on fungal pathogens in respiratory tract infection based on nucleic acid isothermal amplification has important guiding significance for realizing rapid and accurate diagnosis of patients.
Disclosure of Invention
In order to solve the problems, the invention provides a kit for detecting respiratory tract infection fungal pathogens by using an enzyme digestion probe at a constant temperature, wherein 8 pathogen targets are synthesized into three multiple nucleic acid reaction liquids by using respective primer probe sets, and the first multiple nucleic acid reaction liquid comprises primers and probes for detecting candida albicans, aspergillus and candida parapsilosis; the second multiple nucleic acid reaction solution comprises primers and probes for detecting candida glabrata, candida tropicalis and yersinia sporogenes; the third multiple nucleic acid reaction solution comprises primers and probes for detecting mucor and detecting candida krusei; the probe for detecting each fungal pathogen is a probe containing one RNA base and comprises 6 isothermal amplification primers, wherein the fluorescent reporter group of the probe is marked on the left side of the RNA base in the 5 '-3' direction, and the fluorescent quencher group of the probe is marked on the right side of the RNA base in the 5 '-3' direction; the probe for detecting each fungal pathogen in each multiplex nucleic acid reaction solution is labeled with a different fluorophore.
In one embodiment, the first multiplex nucleic acid reaction solution comprises the following primers and probes for detecting candida albicans, aspergillus, and candida parapsilosis, and the primers for detecting candida albicans are SEQ ID nos: 1-6, the probe is SEQ ID No: 7 is shown in the specification; the primers for detecting aspergillus are SEQ ID No: 8-13, the probe is SEQ ID No: 14; and the primer for detecting candida parapsilosis is SEQ ID No: 15-20, the probe is SEQ ID No: 21,
wherein "CA" represents Candida albicans, "Asp" represents Aspergillus, and "Cpa" represents Candida parapsilosis.
In one embodiment, the second multiplex nucleic acid reaction solution comprises the following primers and probes for detecting Candida glabrata, Candida tropicalis and Coilia yarrowia, and the primers for detecting Candida glabrata are SEQ ID Nos: 22-27, the probe is SEQ ID No: 28 is shown; the primer for detecting the candida tropicalis is SEQ ID No: 29-34, the probe is SEQ ID No: 35; and the primer for detecting yersinia sporogenes is SEQ ID No: 36-41, the probe is SEQ ID No: 42,
wherein "Cg" represents Candida glabrata, "Ctr" represents Candida tropicalis, and "PJ" represents Pyezobium yezoensis.
In one embodiment, the third multiplex nucleic acid reaction solution comprises the following primers and probes for detecting mucor and detecting candida krusei, and the primer for detecting mucor is SEQ ID No: 43-48, the probe is SEQ ID No: 49 is shown; the primer for detecting the candida krusei is SEQ ID No: 50-55, the probe is SEQ ID No: 56,
wherein "Muc" represents Mucor, and "Kr" represents Candida krusei.
In one embodiment, the fluorescent reporter group may be any one of FAM, VIC, HEX, ROX, Texas Red, CY3, CY5, respectively, and the quencher group is any one of BHQ1, BHQ2, BHQ 3.
In the prior art, because each target isothermal amplification is designed with six primers and one probe, because of more primers, the realization of multiple isothermal amplification in real detection has great difficulty, and the requirements on the primers and the probes are particularly high. In the invention, under the condition of the optimally designed primers and probes, the 8 fungal pathogens are grouped into 8 fungal pathogens, namely, the 8 fungal pathogens are subjected to multiple isothermal amplifications, namely, the three fungal pathogens are subjected to multiple isothermal amplifications, and the multiple isothermal amplifications are subjected to multiple isothermal amplifications. The plasmid detection of respective 5copies/uL concentration can be realized under the condition of multiple isothermal amplification and the condition of respective isothermal single amplification, and the detection of a plurality of fungal pathogens in each group of reaction liquid in three groups of reaction liquid of the invention has no mutual influence, so that the single-tube multiple detection is carried out in the nucleic acid isothermal amplification method, and the sensitivity and the specificity of the multiple detection reach the technical effect of the single amplification.
In addition, the sensitivity and specificity of multiplex detection achieved in the present invention to that of single amplification are the result of the combined action of the primers and probes of the present invention, and in the case of the same multiplex detection primers of the present invention, the inventors have tried to use a probe of a non-RNA base or to reduce the number of primers per target, and the sensitivity of multiplex detection achieved in the present invention is reduced by one order of magnitude compared to that of single amplification.
Therefore, the nucleic acid detection kit provided by the invention can realize the multiplex isothermal amplification detection of four single tubes for 8 fungal pathogens with high frequency in respiratory tract infection, is simple and convenient to operate, can realize bedside detection, can quickly and accurately screen various fungal pathogens in a sample to be detected within 30min during the whole detection.
Therefore, the invention establishes a nucleic acid isothermal multiple amplification detection respiratory tract infection fungal pathogen, and has important guiding significance for realizing rapid and accurate diagnosis of patients.
Detailed Description
In order to make the technical solutions in the present application better understood, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, but not all of the examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
In the following examples, unless otherwise specified, all methods are conventional in the art. Isothermal amplification buffer, MgSO4, Bst DNA polymerase were obtained from New England Biolabs, N.Y., dNTP was obtained from Bao bioengineering (Dalian) Inc., RNaseH was obtained from American IDT Inc., and primers, probes, and synthetic genes were synthesized by Shanghai Bioengineering services, Inc. The plasmid miniextraction kit and the fungal genome DNA extraction kit are purchased from Beijing Tiangen Biotech limited.
Example one determination of primer Probe combination for multiplex detection of nucleic acids of 8 fungal pathogens of respiratory infection
1. Design of single primer and probe
The invention analyzes the gene sequences of fungal pathogens such as Candida Albicans (CA), candida parapsilosis (Cpa), candida glabrata (Cg), candida tropicalis (Ctr), candida krusei (Kr), aspergillus (Asp), mucor (Muc), yarrowia Pneumocystis (PJ), and the like, designs by using design software on the basis of the basic principle of primer probe design, screens the combination of a primer and a probe aiming at each pathogen target, and the combination of the primer and the probe screened for each pathogen target is shown in a table 1.
TABLE 1.8 primer-probe combinations for screening of fungal pathogens
2. Screening of singleplex primers and probes
2.1 the single weight reaction liquid system is shown in Table 2 below:
TABLE 2 composition table of reagent components of single reaction solution
| Reaction reagent | The quantitative concentration of the final substance or the final enzyme activity unit |
| 10 × isothermal amplification buffer | 1× |
| MgSO4 | 6mM |
| dNTP | 1.4mM |
| Bst DNA polymerase | 8U |
| Betaine solution, PCR grade (5M) | 0.24M |
| RNaseH2 | 15mU |
| Single primer mix | 2.6μM |
| Single probe (20P) | 0.2μM |
| Water (W) | Make up to 30 mu L |
2.2 the templates used were: 8 fungal pathogens low concentration standard plasmid diluent (each 5 copies/L and 10 copies/L two concentrations) and respective genomic DNA, human genomic DNA.
2.3 reaction procedure: 30cycles:63 ℃ for 1min (Collection of fluorescence)
2.4 screening results: as shown in table 3 below, the primer probe combinations with the best sensitivity and specificity were screened for all 8 targets: the primer probe set 1 for CA, the primer probe set 3 for Cpa, the primer probe set 2 for Cg, the primer probe set 2 for Ctr, the primer probe set 1 for Kr, the primer probe set 1 for Asp, the primer probe set 3 for Muc, and the primer probe set 1 for PJ. The optimal primer probe combination of each target can amplify to 5 copies/mu L from the sensitivity point of view, and the primer probe combination with the sensitivity meeting the requirement has no amplification signal when amplifying human and other pathogens, i.e. no cross reaction is generated.
TABLE 3 validation results of screening 8 fungal pathogen primer probe combinations
3. Determination of target combinations in multiplex reaction solutions
Isothermal nucleic acid amplification experiments do not simply mix pairs of specific primer probes into one reaction system. In a constant-temperature multiplex experiment, multiple pairs of primers of each target are required to be not combined with each other, and the primer probe of each target is required to be not combined with a region except a target fragment on a template DNA, so that the specificity in multiplex detection is ensured; in isothermal amplification, the number of primers used for each target is large, so that in a multiplex experiment, the concentration of the primer probes used for a plurality of targets needs to be optimized and adjusted to achieve high amplification sensitivity.
3.1 multiplex detection lines are shown in Table 4 below:
TABLE 4 composition table of reagent components of multiple reaction solutions
| Reaction reagent | Quantitative concentration of end substance or end enzyme activity unit |
| 10 × isothermal amplification buffer | 1× |
| MgSO4 | 6mM |
| dNTP | 1.4mM |
| Bst DNA polymerase | 8U |
| Betaine solution, PCR grade (5M) | 0.24M |
| RNaseH2 | 15mU |
| Primer mix for target 1 | 1.3μM |
| Primer mix for target 2 | 1.3μM |
| Primer mix for target 3 | 1.3μM |
| Target 1 Probe (20P) | 0.2μM |
| Target 2 Probe (20P) | 0.2μM |
| Target 3 Probe (20P) | 0.2μM |
| Water (I) | Make up to 30 mu L |
3.2 reaction procedure: 30cycles:63 ℃ for 1min (Collection of fluorescence)
3.3 determination of target combinations in three multiplex nucleic acid reaction solutions:
the optimal primer probe combinations screened from 8 pathogen targets are subjected to group multiplex verification respectively, as shown in the following table, the amplification effects of different multiplex combinations are different, and the optimal target combinations in three (or two) heavy nucleic acid reaction solutions are verified to be respectively: "CA + Asp + Cpa", "Cg + Ctr + PJ", "Muc + Kr".
TABLE 5.8 Combined screening results for multiple groups of fungal pathogens
From the experimental results in tables 3 and 5 above, it can be seen that in the case of the primers and probes designed according to the present invention, "CA + Asp + Cpa", "Cg + Ctr + PJ" and "Muc + Kr" can achieve plasmid detection at 5copies/uL concentration under the condition of multiplex isothermal amplification and under the condition of isothermal single amplification, respectively, indicating that there is no mutual influence in the detection of three (or two) fungal pathogens in each group of reaction solutions in the three groups of reaction solutions according to the present invention.
In the prior art, because each target isothermal amplification is designed with six primers and one probe, because of more primers, the realization of multiple isothermal amplification in real detection has great difficulty, and the requirement on the primers is particularly high. Eighteen (or twelve) primers and three (or two) enzyme digestion detection probes exist in each reaction tube for detecting three targets, so that detection of each fungal pathogen is not interfered with each other in the primers and the enzyme digestion probes designed by the invention, and the detection sensitivity same as that of single detection is realized.
Meanwhile, the inventor tries to use a detection probe which is not the invention or reduce the number of primers of each target under the condition of the same primer design, and the experimental result shows that the sensitivity is obviously reduced, and the positive plasmid with the detection sensitivity of 100copies/uL concentration can be detected. The designed primers and enzyme digestion probes of the invention realize the detection sensitivity same as that of single detection, on one hand, 18 (or 12) primer pairs in one tube reaction do not interfere with each other during constant temperature diffusion, thus reducing non-specific amplification, and on the other hand, the RNA base probe of the invention is used, thus improving the sensitivity of multiple amplification of the invention.
Example II preparation of the detection kit of the invention and verification of the sensitivity and specificity of the kit
1. Preparation of the kit
Packaging 3 nucleic acid reaction solutions, a blank reference substance and a positive reference substance together, and matching with a product use instruction to obtain the kit for detecting 8 high-fungal pathogens in respiratory tract infection at constant temperature by using the enzyme digestion probe. The compositions of the nucleic acid reaction solution, the blank control and the positive control are shown in the following table 6, and the fluorescent group labels of the probes for detecting each pathogen are shown in the following table 7.
TABLE 6 composition table of kit components
TABLE 7 fluorescent group labeling instruction table for each probe in the kit
2. The kit of the invention detects 4 clinical samples
2.1 samples used were: s1 (Candida albicans positive clinical specimen DNA for respiratory infection), S2 (Candida parapsilosis positive clinical specimen DNA for respiratory infection), S3 (Yersinia pneumonoconioides positive clinical specimen DNA for respiratory infection), and S4 (genome DNA extracted from clinical specimen negative for respiratory infection).
2.2 Using the prepared kit, 3 nucleic acid reaction solutions were detected for each sample, and the loading volume of each nucleic acid reaction solution was 10. mu.L.
2.3 reaction procedure: 30cycles:63 ℃ for 1min (Collection of fluorescence)
3. The detection results are shown in the following table 8, S1 shows that there is an amplification signal in the FAM channel, no amplification signal in the Cy5 channel and the ROX channel, and no amplification signal in the other 2 nucleic acid reaction solutions, and it can be determined that candida albicans infection is positive; s2 shows that the CY5 channel in the nucleic acid reaction solution 1 has amplification signals, the FAM channel and the ROX channel have no amplification signals, and other 2 nucleic acid reaction solutions have no amplification signals, so that the positive candida parapsilosis infection can be judged; s3 shows that there is amplification signal in CY5 channel in the nucleic acid reaction solution 2, there is no amplification signal in FAM channel and ROX channel, there is no amplification signal in other 2 nucleic acid reaction solutions, and it can be judged that the infection of Yersinia pneumocystis is positive; s4 shows no amplification signal in all 3 nucleic acid reaction solutions, and it was judged that these 8 fungal pathogens were not infected. The interpretation of each sample was consistent with the actual infection of the sample.
TABLE 8 results of 4 clinical specimens tested by the kit of the present invention
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Sequence listing
<110> general hospital of liberation military of Chinese people
BEIJING MACRO & MICRO TEST BIO TECH Co.,Ltd.
JIANGSU MACRO & MICRO-TEST PHARMACEUTICAL TECHNOLOGY Co.,Ltd.
<120> enzyme digestion probe constant temperature detection respiratory tract infection fungal pathogen kit
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
gtctgatgtg ccaaatgg 18
<210> 55
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gattttgtta tcggaaggcc 20
<210> 56
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
aacattgttt cgggttctat gtctg 25
Claims (2)
1. A kit for detecting respiratory tract infection fungal pathogens at constant temperature by using an enzyme digestion probe is characterized in that 8 pathogen targets are synthesized into three multiple nucleic acid reaction liquids by using respective primer probe sets, wherein the first multiple nucleic acid reaction liquid comprises primers and probes for detecting Candida albicans, Aspergillus and Candida parapsilosis; the second multiple nucleic acid reaction solution comprises primers and probes for detecting candida glabrata, candida tropicalis and yersinia pneumocystis; the third multiple nucleic acid reaction solution comprises primers and probes for detecting mucor and detecting candida krusei;
the probe and the primer for detecting each fungal pathogen are probes containing one RNA base and comprise 6 isothermal amplification primers, wherein the fluorescent reporter group of the probe is marked on the left side of the RNA base in the 5 '-3' direction, and the fluorescent quencher group of the probe is marked on the right side of the RNA base in the 5 '-3' direction; labeling different fluorescent groups with probes for detecting each fungal pathogen in each multiplex nucleic acid reaction solution;
the first multiplex nucleic acid reaction solution comprises the following primers and probes for detecting candida albicans, aspergillus and candida parapsilosis, wherein the primers for detecting candida albicans are SEQ ID No: 1-6, the probe is SEQ ID No: 7 is shown in the specification; the primers for detecting aspergillus are SEQ ID No: 8-13, the probe is SEQ ID No: 14; and the primer for detecting candida parapsilosis is SEQ ID No: 15-20, the probe is SEQ ID No: 21,
wherein "CA" represents Candida albicans, "Asp" represents Aspergillus, and "Cpa" represents Candida parapsilosis, the underlined bases in italics indicate RNA bases; the second multiplex nucleic acid reaction solution comprises the following primers and probes for detecting candida glabrata, candida tropicalis and yersinia sporogenes, wherein the primers for detecting candida glabrata are SEQ ID No: 22-27, the probe is SEQ ID No: 28 is shown; the primer for detecting the candida tropicalis is SEQ ID No: 29-34, the probe is SEQ ID No: 35; and the primer for detecting yersinia sporogenes is SEQ ID No: 36-41, the probe is SEQ ID No: 42,
where "Muc" represents Mucor, and "Kr" represents Candida krusei, and the underlined bases in italics indicate RNA bases.
2. The kit according to claim 1, wherein the fluorescent reporter group is any one of FAM, VIC, HEX, ROX, Texas Red, CY3 and CY5, and the quencher group is any one of BHQ1, BHQ2 and BHQ 3.
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| CN202110078008.XA CN112626263B (en) | 2021-01-20 | 2021-01-20 | Kit for detecting respiratory tract infection fungal pathogens at constant temperature by using enzyme digestion probe |
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| CN202110078008.XA CN112626263B (en) | 2021-01-20 | 2021-01-20 | Kit for detecting respiratory tract infection fungal pathogens at constant temperature by using enzyme digestion probe |
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| CN112626263B true CN112626263B (en) | 2022-08-05 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| ES2621547T3 (en) * | 1999-05-28 | 2017-07-04 | Fujirebio Europe N.V. | Nucleic acid probes and methods for detecting clinically important fungal pathogens |
| US20050227236A1 (en) * | 2003-02-10 | 2005-10-13 | Forsberg Neil E | Mold infections |
| JP2010532665A (en) * | 2007-07-11 | 2010-10-14 | ユニヴェルシテ ラヴァル | Nucleic acid sequences and their combinations for sensitive amplification and detection of bacterial and fungal sepsis pathogens |
| GB2561555A (en) * | 2017-04-12 | 2018-10-24 | Momentum Bioscience Ltd | Detection and delineation of microorganisms |
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