CN110616253A - Kit and method for detecting fungi based on micro-drop digital PCR technology - Google Patents
Kit and method for detecting fungi based on micro-drop digital PCR technology Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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
Abstract
The invention provides a kit for detecting fungi based on a digital PCR technology, which comprises: (1) a forward primer, a reverse primer, a probe (2) buffer solution and a mixed reagent of PCR amplification enzyme; (3) extracting reagent for fungus genome DNA; (4) positive and negative controls; (5) RNase-free ultrapure water. On the basis of the kit, the invention also provides a method for detecting fungi based on the digital PCR technology. Experiments prove that the kit provided by the invention has the advantages of good specificity, high sensitivity and good repeatability, and the established method for detecting the fungi based on the digital PCR technology is sensitive, accurate, simple, convenient and quick, and has guiding significance for qualitative and quantitative detection of the fungi.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a kit and a method for detecting fungi based on a droplet type digital PCR technology.
Background
The current gold standard for mycology diagnosis is culture positive of blood or cerebrospinal fluid or discovery of fungi under a microscope. Although the fungus culture can clearly identify the pathogenic bacteria infected by the fungus and carry out the in vitro drug sensitivity test, the positive rate of the blood culture is low. 1, 3-beta-D-glucan is a fungal cell wall component that has been used to aid in the diagnosis of various fungal infections. Factors such as gauze, cotton balls, some antibiotic drugs, intravenous albumin or gamma globulin, plasma, tissue thromboplastin, etc. can sometimes cause the 1, 3-beta-D-glucan test (G test) to show false positive results. Galactomannan antigens are main components of aspergillus cell walls and are released by hyphae in the growth process of aspergillus, and a galactomannan antigen detection (GM) test mainly aims at aspergillus infection, but GM test results are also influenced by a plurality of factors, such as piperacillin sodium/tazobactam and the use of a large dose of hormone can cause false positive.
Eukaryotic microorganisms have three classes of ribosomal RNA (rRNA), including 5.8S rRNA, 18S rRNA, and 28S rRNA. The 18S rRNA gene is a DNA sequence encoding the small subunit of the eukaryotic ribosome, which has both conserved and variable regions. The conserved regions reflect the relationship between biological species, while the variable regions reflect species-to-species differences. The biological method for detecting fungi by utilizing the 18S rRNA conserved region has the conventional PCR technology and the real-time fluorescent quantitative PCR technology, the sensitivity of the technologies can be equal to or higher than that of blood culture, and the conventional PCR technology and the real-time fluorescent quantitative PCR technology have inherent limitations, such as the preparation of a standard product, the quantification through a standard curve and the like, and the accurate and precise quantification of the pathogen load capacity can not be realized.
Disclosure of Invention
The invention aims to solve the technical problem of providing a kit and a method for detecting candida tropicalis based on a micro-drop digital PCR (ddPCR) technology, wherein the kit provided by the invention has the advantages of good specificity, high sensitivity and good repeatability, so that the established method for detecting fungi based on the micro-drop digital PCR technology is sensitive, accurate, simple, convenient and quick, and has guiding significance for qualitative and quantitative detection of fungi.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention provides a kit for detecting fungi based on a droplet type digital PCR technology, which is characterized by comprising: (1) a forward primer: 5'-GTGAATCATCGAATCTTTGAAC-3', respectively; reverse primer: 5'-TCCTCCGCTTATTGATATGC-3', respectively; and (3) probe: 5 '-FAM-ATTGCTTGCGGCGGTAACGTCC-TRAMA-3', wherein the 5-end labeled fluorophore is FAM and the 3-end labeled quencher is TRAMA; (2) buffer solution and PCR amplification enzyme mixed reagent; (3) extracting reagent for fungus genome DNA; (4) positive and negative controls; (5) RNase-free ultrapure water.
Preferably, the buffer and the PCR amplification enzyme mixed reagent are 2 XddPCR Supermix for Probes reagent of product No. 1863010 of Bio-Rad company; the fungal genome DNA extraction reagent is a reagent contained in a genome extraction Kit QIAamp UCP Pathogen Mini Kit of QIAGEN company in Germany; the concentrations of the upstream primer, the downstream primer and the probe are respectively 25pmol/ul, 25pmol/ul and 100pmol/ul, and the molar concentration ratio of the upstream primer, the downstream primer and the probe is 1:1:4 when the amplification reaction is carried out.
Preferably, the positive control is fungal genomic DNA or a nucleic acid sequence containing fungal 18S rRNA reverse transcription DNA, and the negative control is bacterial DNA. The nucleic acid sequence comprises a plasmid containing fungal 18S rRNA reverse transcribed DNA.
In another aspect of the present invention, there is provided a method for detecting fungi based on the digital PCR technique in microdroplet form, the method being used for non-diagnostic purposes, the method comprising the steps of:
the method comprises the following steps: extraction of genomic DNA
Extracting fungal genome DNA in a sample to be detected by using an extraction reagent in a genome extraction Kit QIAamp UCP Pathologen Mini Kit of Germany QIAGEN company and an extraction method thereof;
step two: a droplet-based digital PCR amplification, comprising the steps of:
(1) preparing a reaction system: respectively adding 10 mu L of 2 XddPCR Supermix for Probes with product number 1863010 of Bio-Rad company into each reaction tube of a sample to be detected, a negative control and a positive control, wherein the final concentration of an upstream primer and a downstream primer is 25pmol/L, the final concentration of a probe is 100pmol/L, and the DNA template is 2 ul; supplementing the volume to 20 mu L by ultrapure water, wherein the template DNA comprises the DNA of a sample to be detected, a negative control and a positive control;
(2) preparing microdroplets: transferring the prepared 20 mu L of PCR reaction solution to a microdroplet generation clamping hole, and then adding 70 mu L of microdroplet generation oil into an oil hole to prepare microdroplets;
(3) amplification: transferring the microdroplets into a PCR plate, sealing the plate at 180 ℃ for 5-10 s, and carrying out amplification in a PCR instrument;
step three: signal detection of droplet digital PCR products
And (4) putting the PCR plate amplified in the step two into a microdroplet analyzer, and detecting the fluorescent signal in the microdroplet.
Preferably, the amplification procedure in the above method is pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30s, annealing at 59.2 ℃ for 60s, for 40 cycles; 10min at 98 ℃. Some non-specific amplification and some non-specific fluorescence signals occur more or less in PCR amplification, and in order to reduce interference of the non-specific fluorescence signals and improve detection accuracy, the specificity of fluorescence and a probe needs to be improved. Experiments show that the annealing temperature of 59.2 ℃ is used for amplification, positive microspheres and negative microspheres can be effectively distinguished, namely, the annealing temperature of 59.2 ℃ is used for amplification, primers and probes have high specificity, and whether fungi exist or not and the quantity of the fungi can be effectively identified.
Preferably, the step (1) further comprises a step of quantifying nucleic acid from the genomic DNA by ultraviolet spectrophotometry. The purpose of genomic DNA quantification is to select suitable concentrations of genomic DNA for droplet preparation.
Preferably, the above method further comprises step (5): and (3) judging the result, analyzing and processing the detected fluorescence signal by using QuantaSoft1.7.4 software, wherein the microspheres with the amplitude of more than 3500 are positive microspheres, and the microspheres with the amplitude of less than 3500 are negative microspheres. The positive microspheres mean that the microspheres contain fungal DNA, and the negative microspheres mean that the microspheres do not contain fungal DNA.
The invention has the following beneficial effects:
1) the invention provides a detection kit aiming at fungi, establishes a method for detecting the fungi based on a micro-drop digital PCR technology, and can be used for qualitatively and quantitatively detecting the fungi.
2) When the kit and the method for detecting the fungi based on the micro-drop digital PCR technology are used for detection, the lower detectable limit is 3.8 copies/mu L (copy/mu L), so that the fungi and the bacteria can be effectively distinguished; and the coefficient of variation for both the inter-and intra-batch replicates was less than 3%.
3) The kit for detecting the fungi based on the micro-drop digital PCR technology has the advantages of good detection specificity, high sensitivity, good repeatability and wide practical range.
4) The method for detecting the fungi based on the micro-drop digital PCR is sensitive, accurate, simple, convenient and quick, and has guiding significance for qualitative and quantitative detection of the fungi.
Description of the drawings:
FIG. 1: FIG. 1-a shows a one-dimensional scattergram of Candida tropicalis genomic DNA amplified by a droplet digital PCR method gradient temperature; FIG. 1-b shows a peak at an optimum temperature of 59.2 ℃.
FIG. 2: FIG. 2-a shows a one-dimensional scattergram of Candida tropicalis genomic DNA amplified by a droplet-based digital PCR method with gradient dilution; FIG. 2-b shows the correlation analysis chart of the detection result of the plasmid ddPCR constructed by fungal 18sRNA and the theoretical value.
FIG. 3: shows a one-dimensional scatter diagram of the micro-drop digital PCR method for gradient amplification of 3 strains of fungal genomic DNA and 10 strains of bacterial genomic DNA. The scatter above the horizontal line represents positive droplets and the scatter below the horizontal line represents negative droplets, wherein 3 strains of fungal genomic DNA see a large number of positive droplets, while 10 strains of bacterial genomic DNA do not see positive droplets. The specific names in the figures are represented as follows: tr: candida tropicalis; c.pa: candida parapsilosis 2.3989; sm: candida glabrata 2.3989; pae: pseudomonas aeruginosa; cma: corynebacterium glutamicum; eae: aerogenic bacillus; ec: enterobacter cloacae; sa: staphylococcus aureus bacteria; GBs: group B streptococci; and GAs: group a streptococcus; coli: e.coli; kpn: klebsiella pneumoniae; aba: acinetobacter baumannii.
Detailed Description
Unless otherwise defined, the terms used herein have the ordinary meanings as commonly understood in the art to which this invention belongs.
The present invention will now be described with reference to specific examples and figures, which are provided for illustrative purposes only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are in accordance with routine experimentation or with the manufacturer's instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The invention relates to the following main instruments:
ND-1000 Spectrophotometer: nanodrop, usa;
c1000touch PCR instrument, QX200TM ddPCR system: Bio-Rad, USA;
QIAamp UCP Pathologen Mini Kit and Pathologen lysine Tubes: QIAGEN, germany;
the invention relates to the following main reagent materials:
table 1 lists the strains selected in the examples: 3 Candida strains (Candida tropicalis, Candida parapsilosis 2.3989, Candida glabrata 2.3983) and 10 non-fungal strains.
TABLE 1 strains and sources
| Bacterial strains | Origin of origin |
| Candida tropicalis | China general microbiological culture preservation management center |
| Candida parapsilosis 2.3989 | China general microbiological culture preservation management center |
| Candida glabrata 2.3983 | China general microbiological culture preservation management center |
| Klebsiella pneumoniae | Southern hospital microbial room of southern medical university |
| Acinetobacter baumannii | Southern hospital microbial room of southern medical university |
| Group B streptococcus | American type culture Collection |
| Group A streptococcus | Southern hospital microbial room of southern medical university |
| Pseudomonas aeruginosa | Southern hospital microbial room of southern medical university |
| Aerobacter aerogenes | Southern hospital microbial room of southern medical university |
| Enterobacter cloacae | Southern hospital microbial room of southern medical university |
| Staphylococcus aureus | Southern hospital microbial room of southern medical university |
| Escherichia coli | Southern hospital microbial room of southern medical university |
| Corynebacterium glutamicum | Southern hospital microbial room of southern medical university |
Example 1
This example provides sequences and uses of primer and probe combinations:
the screening method of the combination of the primer and the probe comprises the following steps: aiming at the highly conserved 18S rRNA gene sequence of the fungus, a plurality of primers and probes are designed and screened in a large quantity, the specificity, the sensitivity, the functions between the primers and the probes and the adaptability of each primer and a digital PCR amplification kit are synthesized, and finally the following combinations of the primers and the probes which can detect the fungus, have good specificity, good repeatability and high sensitivity are screened out:
forward primer (5 '-3'): GTGAATCATCGAATCTTTGAAC, respectively;
reverse primer (5 '-3'): TCCTCCGCTTATTGATATGC, respectively;
probe (5 '-3'): FAM-ATTGCTTGCGGCGGTAACGTCC-TRAMA.
The probe is a TaqMan probe and is positioned in a region between primers, FAM is used as a report dye for carrying out fluorescent labeling on the 5 'end of the probe, TRAMA is used as a quencher on the 3' end of the probe, and the primers and the TaqMan probe are synthesized by Shanghai Czeri bioengineering GmbH.
In application, the primers and the probes can be used for preparing products for detecting fungi based on a digital PCR technology, and can be directly used for detecting or assisting in detecting whether the fungi exist in a sample.
Example 2
This embodiment provides a kit and its application, the kit includes:
(1) an upstream primer shown as SEQ ID NO. 1, a downstream primer shown as SEQ ID NO. 2 and a probe shown as SEQ ID NO. 3;
(2) a mixed reagent of buffer solution and PCR amplification enzyme;
(3) extracting reagent for fungus genome DNA;
(4) positive and negative controls;
(5) rnase-free ultrapure water;
the mixed reagent of the buffer and the enzyme is 2 XddPCR Supermix for Probes reagent of product No. 1863010 from Bio-Rad company.
The fungal genomic DNA extraction reagent is a reagent contained in a genomic extraction Kit QIAamp UCP Pathogen Mini Kit from QIAGEN, Germany.
The positive control is fungal genome DNA or a nucleic acid sequence containing fungal 18S rRNA reverse transcription DNA, preferably Candida tropicalis genome DNA, and the negative control is bacterial DNA.
Experiments prove that the upstream primer shown in SEQ ID NO. 1, the downstream primer shown in SEQ ID NO. 2, the probe shown in SEQ ID NO. 3, a mixed reagent of a buffer solution and a PCR (polymerase chain reaction) amplification enzyme and a fungus genome DNA extraction reagent are jointly applicable, the effect is more excellent, the specificity is strong, the repeatability is good, the sensitivity is high, and the method can be used for accurately qualitatively and quantitatively analyzing fungi.
In application, the kit can be used for detecting or assisting in detecting fungi, for example, detecting or assisting in detecting whether a sample to be detected contains the fungi and the loading capacity of the fungi.
Example 3
This example provides a method for detecting fungi based on the digital PCR technique of microdroplet, which is used for non-disease detection and diagnostic purposes. The method uses the primer and probe combination of example 1 or the kit of example 2.
The method for detecting fungi based on the micro-drop digital PCR technology provided by the embodiment comprises the following steps:
the method comprises the following steps: extraction of genomic DNA
Extracting fungal genome DNA: after enrichment culture of fungi in a Sasa liquid medium, genomic DNA of the fungi was extracted using the QIAamp UCP pathway Mini Kit and pathway lysine Tubes (QIAGEN, Germany), and the genomic DNA was obtained by subsequent manipulation according to the fungal DNA extraction method proposed in the manual of the QIAamp UCP pathway Mini Kit.
Then, the extracted DNA is quantified by an ultraviolet spectrophotometry, the DNA copy number concentration (copy/. mu.L) is calculated according to the molecular weight of the DNA, and the DNA is diluted to a proper concentration to be configured into a digital PCR reaction system. The DNA concentration is chosen to be below 20000 copies/. mu.l and above 1 copy/. mu.l. The concentration was chosen according to the sensitivity detection experiment and the established drop saturation curve in example 4. The DNA which is not used for the subsequent use is stored at-20 ℃ for later use.
Step two: droplet digital PCR amplification
(1) Preparing a reaction system: 10 μ L2 XDdPCRTMSupermix for Probes (Bio-Rad, product No. 1863010), final concentrations of 25pmol/ul for forward and reverse primers, 100pmol/ul for probe, and 2ul for DNA template, and made up to 20. mu.L with RNase-free ultrapure water.
(2) Preparing microdroplets: transferring 20 μ L of the prepared PCR reaction solution to a microdroplet generation card well, adding 70 μ L of microdroplet generation oil (Bio-rad) to an oil well, and performing quantitative analysis by using QX200TMThe microdroplet generator of the ddPCR system prepares reaction microdroplets.
(3) Amplification: transferring the reaction microdroplets of each sample to corresponding reaction holes of a 96-hole PCR reaction plate, performing heat sealing (at 180 ℃ for 5s) by using an aluminum film, and performing amplification on a common PCR instrument, wherein the PCR amplification is completed on a C1000touch PCR instrument.
The annealing temperature is optimized through gradient PCR, and finally the selected ddPCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30s, annealing at 59.2 ℃ for 60s, for 40 cycles; after the amplification, thermal denaturation at 98 ℃ was carried out for 10 min. Each reaction well was replicated 3 times for parallel detection.
Step three: signal detection of droplet digital PCR products
Putting the 96-well plate after PCR amplification into QX200TMIn a droplet analyzer of ddPCR system (normal temperature), fluorescence signal of FAM is detected, and then automatic processing of data is completed by QuantaSoft1.7.4 software.
And (5) judging a result:
by taking Candida tropicalis genome DNA as a template, positive droplets and negative droplets of the Candida tropicalis are most separated at 59.2 ℃, the number of the droplets in the blank between the droplets is minimum (figure 1-a), a peak diagram shows that the positive peaks and the negative peaks are obviously separated at 59.2 ℃, and no interference exists in the middle (figure 1-b), and the ddPCR probe concentration and the amplification system determined by optimization are very suitable for quantitative analysis of fungi. The dots above the horizontal line in FIG. 1-a represent positive droplets and the dots below the horizontal line represent negative droplets. The positive and negative droplets are distinguished at different temperatures. In FIG. 1-b, the left side shows a negative peak (negative droplet peak) and the right side shows a positive peak (positive droplet peak), which are significantly separated and have no interference in the middle. The horizontal line in FIG. 1-a/vertical line in FIG. 1-b is the median of the low amplitude of the positive droplets and the high amplitude of the negative droplets at 59.2 ℃ when the positive droplets and the negative droplets are most separated.
As can be seen from the above, in the kit provided in example 2 of the present invention and the method for detecting fungi by the droplet-type digital PCR technique using the kit according to this example, qualitative and quantitative analysis of fungi can be performed very efficiently without interference by using various reagents in the kit, and parameters such as the primer, probe concentration, and amplification system program related to the method in combination.
Example 4
This example performed sensitivity verification of the primer and probe combination of example 1 and the kit of example 2 using the biological materials and sources shown in Table 1.
The sensitivity verification experimental procedure is as follows:
the method comprises the following steps: genomic DNA extraction
Genomic DNA of Candida tropicalis was extracted in the same manner as in the step of example 3.
Step two: sensitivity detection
And (3) carrying out 10-fold gradient dilution on the genomic DNA extracted in the step one to prepare genomic DNAs of the candida tropicalis with different concentrations, and respectively carrying out droplet type digital PCR amplification and signal detection by using the genomic DNAs of the candida tropicalis with different concentrations as templates, wherein the method is the same as the droplet type digital PCR amplification in the step two and the signal detection of the droplet type digital PCR product in the step three in the embodiment 3.
And (4) analyzing results:
the results are shown in FIG. 2, and it can be seen from FIG. 2 that the primer and probe combination and kit of the present invention can detect the DNA fragment 106Candida tropicalis genomic DNA was diluted in multiples with a detection limit of 3.8 copies/. mu.L (3.8 copies/. mu.L). FIG. 2-a is a diagram of a droplet-type digital PCR method gradientAnd (3) diluting and amplifying the Candida tropicalis genome DNA one-dimensional scatter diagram. When 10-fold gradient dilution is carried out, the positive droplets are reduced in gradient until the dilution factor is 107When no positive droplets are detected.
Droplet saturation curves:
ddPCR the copy number concentration of the 18S rRNA-constructed plasmid and the corresponding copy number measured using ddPCR were fitted to a linear regression model using Microsoft Excel software (Microsoft, USA) (FIG. 2-b). The ddPCR detection is in a dynamic range (the plasmid concentration is 1.5 multiplied by 10-1.5 multiplied by 10)4Copies/. mu.l) are in a highly linear relationship. The value of r is very close to 1(r ═ 0.997). When the target concentration is more than or equal to 105At copy/ul, positive saturation of the droplets was observed.
Example 5
This example demonstrates the specificity of the primer and probe combination of example 1 and the kit of example 2 using two methods, the biological materials and sources used are shown in Table 1.
The method comprises the following steps: the primers and probes of example 1 were used to perform ddPCR reactions on the three fungal strains of Table 1, and the reaction products were sequenced. The results obtained from sequencing showed 100% homology to the sequence of the fungal 18S rRNA gene in the GenBank database.
The second method comprises the following steps: microdroplet digital PCR
After 3 strains of fungi and 10 strains of bacteria (table 1) are subjected to enrichment culture, corresponding genomic DNA is extracted, and all DNAs are simultaneously detected by adopting an optimized ddPCR detection method.
The experimental procedure for verifying specificity by microdroplet digital PCR was as follows:
the method comprises the following steps: genome extraction
(1) Extracting fungal genome DNA: after enrichment culture of fungi in a Sasa liquid medium, genomic DNA of the fungi was extracted using the QIAamp UCP pathway Mini Kit and pathway lysine Tubes (QIAGEN, Germany), and the genomic DNA was obtained by subsequent manipulation according to the fungal DNA extraction method proposed in the manual of the QIAamp UCP pathway Mini Kit.
(2) Extracting bacterial genome DNA: after various bacteria were cultured in the corresponding medium for enrichment, the bacterial genomic DNA was extracted with the QIAamp DNA blood Mini Kit (QIAGEN, Germany), and the subsequent operations were performed according to the method for extracting bacterial DNA suggested in the manual of the QIAamp DNA blood Mini Kit to obtain the genomic DNA.
Then, the extracted DNA is quantified by an ultraviolet spectrophotometry, the DNA copy number concentration (copy/. mu.L) is calculated according to the molecular weight of the DNA, and the DNA is diluted to a proper concentration to be configured into a digital PCR reaction system. The DNA concentration is chosen to be below 20000copy/ul and above 1 copy/ul. The concentration was chosen according to the sensitivity detection experiment and the established drop saturation curve in example 4. The DNA which is not used for the subsequent use is stored at-20 ℃ for later use.
Step two: droplet digital PCR amplification
(1) Preparing a reaction system: 10 μ L2 XDdPCRTMSupermix for Probes (Bio-Rad, product No. 1863010), 25pmol/ul for each of the forward and reverse primer, 200pmol/ul for the probe, and 2ul for the DNA template, and the volume was made up to 20. mu.L with RNase-free ultrapure water.
(2) Preparing microdroplets: transferring 20 μ L of the prepared PCR reaction solution to a microdroplet generation card well, adding 70 μ L of microdroplet generation oil (Bio-rad) to an oil well, and performing quantitative analysis by using QX200TMThe microdroplet generator of the ddPCR system prepares reaction microdroplets.
(3) Amplification: transferring the reaction microdroplets of each sample to corresponding reaction holes of a 96-hole PCR reaction plate, performing heat sealing (at 180 ℃ for 5s) by using an aluminum film, and performing amplification on a common PCR instrument, wherein the PCR amplification is completed on a C1000touch PCR instrument.
The annealing temperature is optimized through gradient PCR, and finally the selected ddPCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30s, annealing at 59.2 ℃ for 60s, for 40 cycles; after the amplification, thermal denaturation at 98 ℃ was carried out for 10 min. Each reaction well was replicated 3 times for parallel detection.
Step three: signal detection of droplet digital PCR products
Putting the 96-well plate after PCR amplification into QX200TMIn a droplet analyzer of the ddPCR system, fluorescence signals of FAM are detected at normal temperature, and then logarithm is completed by QuantaSoft1.7.4 softwareAnd (6) automatically processing.
And (4) analyzing results:
the detection results are shown in FIG. 3, and it can be seen from FIG. 3 that a large number of positive droplets are observed in the ddPCR detection results of 3 strains of fungal strains, while no positive droplets are observed in the ddPCR detection results of 10 strains of bacterial strains. In conclusion, all the amplification internal standards of all groups are successfully amplified, and fungi and bacteria can be effectively distinguished, so that the combination of the primers and the probes in the embodiment 1 and the kit in the embodiment 2 have high specificity; furthermore, the detection method established based on the combination of the primers and the probes can effectively and accurately detect the fungi. In fig. 3, the dots above the horizontal line represent positive droplets and the dots above the horizontal line represent negative droplets. A large number of positive microdroplets were visible for the genomic DNA of 3 strains of fungi. No positive droplets were observed for genomic DNA of 10 strains of bacteria. Tr: candida tropicalis; c.pa: candida parapsilosis; sm: candida glabrata; pae: pseudomonas aeruginosa; cma: corynebacterium glutamicum; eae: aerogenic bacillus; ec: enterobacter cloacae; sa: staphylococcus aureus bacteria; GBs: group B streptococci; and GAs: group a streptococcus; coli: e.coli; kpn: klebsiella pneumoniae; aba: acinetobacter baumannii.
Example 6
This example performed stability verification (reproducibility test) of the primer and probe combination of example 1 and the kit of example 2, and the used biological materials and sources are shown in table 1.
The experimental procedure for stability verification in this example is as follows:
the method comprises the following steps: extraction of genomic DNA
Extracting fungal genome DNA: after enrichment culture of fungi in a Sasa liquid medium, genomic DNA of the fungi was extracted using the QIAamp UCP pathway Mini Kit and pathway lysine Tubes (QIAGEN, Germany), and the genomic DNA was obtained by subsequent manipulation according to the fungal DNA extraction method proposed in the manual of the QIAamp UCP pathway Mini Kit.
Then, the extracted DNA is quantified by an ultraviolet spectrophotometry, the DNA copy number concentration (copy/. mu.L) is calculated according to the molecular weight of the DNA, and the DNA is diluted to a proper concentration to be configured into a digital PCR reaction system. The DNA concentration is chosen to be below 20000 copies/. mu.l and above 1 copy/. mu.l. The concentration was chosen according to the sensitivity detection experiment and the established drop saturation curve in example 4. The DNA which is not used for the subsequent use is stored at-20 ℃ for later use.
Step two: droplet digital PCR amplification
(1) Preparing a reaction system: 10 μ L2 XDdPCRTMSupermix for Probes (Bio-Rad, product No. 1863010), forward and reverse primers, respectively, at a final concentration of 25pmol/ul, at a final concentration of 200pmol/ul, and at a final concentration of 2ul DNA template, were made up to a volume of 20. mu.L with RNase-free ultrapure water.
And (3) carrying out repeated detection, wherein the repeated experiments in batches and among batches all use Candida tropicalis genome DNA as a template to carry out 3 ddPCR detections with different concentrations. The batch-to-batch reproducibility experiment was repeated for the same sample under the same conditions for 3 consecutive days (3 days).
(2) Preparing microdroplets: transferring 20 μ L of prepared PCR reaction solution to microdroplet generation card hole, adding 70 μ L of microdroplet generation oil (BIORAD) to oil hole, and applying QX200TMThe microdroplet generator of the ddPCR system prepares reaction microdroplets.
(3) Amplification: transferring the reaction microdroplets of each sample to corresponding reaction holes of a 96-hole PCR reaction plate, performing heat sealing (at 180 ℃ for 5s) by using an aluminum film, and performing amplification on a common PCR instrument, wherein the PCR amplification is completed on a C1000touch PCR instrument.
The annealing temperature is optimized through gradient PCR, and finally the selected ddPCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30s, annealing at 59.2 ℃ for 60s, for 40 cycles; after the amplification, thermal denaturation at 98 ℃ was carried out for 10 min. Each reaction well was replicated 3 times for parallel detection.
Step three: signal detection of droplet digital PCR products
Putting the 96-well plate after PCR amplification into QX200TMIn a droplet analyzer of the ddPCR system, fluorescence signals of FAM are detected at normal temperature, and then automatic processing of data is completed by QuantaSOFT1.7.4 software.
And (4) analyzing results:
the specific detection results are shown in tables 2 and 3, and the results show that the number of positive micro-drops among the reactions of the samples with the same concentration is close, and the variation coefficients are 1.00%, 2.73% and 2.42% respectively (table 2); the in-batch repeatability test is to perform 3 times of repeated determination on the same sample in the same reaction system, and the result shows that the number of positive microdroplets is close to that of the reaction of the template with the same concentration, and the variation coefficients are respectively 2.82%, 2.24% and 2.71% (table 3), which shows that the primer and probe combination, the kit and the method for detecting the fungi based on the microdroplet digital PCR technology have very good repeatability.
TABLE 2 Candida tropicalis genomic DNA batch-to-batch reproducibility test results
Note: CV: the coefficient of variation.
TABLE 3 results of in-batch reproducibility test of Candida tropicalis genomic DNA
Note: CV: the coefficient of variation.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. For those skilled in the art to which the present application pertains, several simple deductions or substitutions may be made without departing from the concept of the present application, and all should be considered as belonging to the protection scope of the present application.
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Claims (7)
1. A kit for detecting fungi based on a droplet type digital PCR technology is characterized by comprising:
(1) a forward primer: 5'-GTGAATCATCGAATCTTTGAAC-3', respectively;
reverse primer: 5'-TCCTCCGCTTATTGATATGC-3', respectively;
and (3) probe: 5 '-FAM-ATTGCTTGCGGCGGTAACGTCC-TRAMA-3', wherein the 5-end labeled fluorophore is FAM and the 3-end labeled quencher is TRAMA;
(2) a mixed reagent of buffer solution and PCR amplification enzyme;
(3) extracting reagent for fungus genome DNA;
(4) positive and negative controls;
(5) RNase-free ultrapure water.
2. The kit of claim 1, wherein the mixed reagent of the buffer and the PCR amplification enzyme is 2 XDddPCR Supermix for Probes reagent of Bio-Rad company product No. 1863010; the fungal genome DNA extraction reagent is a reagent contained in a genome extraction Kit QIAamp UCP Pathogen Mini Kit of QIAGEN company in Germany; the concentrations of the upstream primer, the downstream primer and the probe are respectively 25pmol/ul, 25pmol/ul and 100pmol/ul, and the molar concentration ratio of the upstream primer, the downstream primer and the probe is 1:1:4 when the amplification reaction is carried out.
3. The kit of claim 1, wherein the positive control is fungal genomic DNA or a nucleic acid sequence comprising fungal 18S rRNA reverse transcribed DNA and the negative control is bacterial DNA.
4. A method for detecting fungi based on a droplet type digital PCR technology is characterized by comprising the following steps:
the method comprises the following steps: extraction of genomic DNA
Extracting fungal genome DNA in a sample to be detected by using an extraction reagent in a genome extraction Kit QIAamp UCP Pathologen Mini Kit of Germany QIAGEN company and an extraction method thereof;
step two: a droplet-based digital PCR amplification, comprising the steps of:
(1) preparing a reaction system: respectively adding 10 mu L of 2 XddPCR Supermix for Probes with product number 1863010 of Bio-Rad company into each reaction tube of a sample to be detected, a negative control and a positive control, wherein the final concentration of an upstream primer and a downstream primer is 25pmol/ul, the final concentration of a probe is 100pmol/ul, and the final concentration of a DNA template is 2 ul; supplementing the volume to 20 mu L by ultrapure water, wherein the template DNA comprises the DNA of a sample to be detected, a negative control and a positive control;
(2) preparing microdroplets: transferring the prepared 20 mu L of PCR reaction solution to a microdroplet generation clamping hole, and then adding 70 mu L of microdroplet generation oil into an oil hole to prepare microdroplets;
(3) amplification: transferring the microdroplets into a PCR plate, sealing the plate at 180 ℃ for 5-10 s, and carrying out amplification in a PCR instrument;
step three: signal detection of droplet digital PCR products
And (4) putting the PCR plate amplified in the step two into a microdroplet analyzer, and detecting the fluorescent signal in the microdroplet.
5. The method of claim 4, wherein the procedure for amplification is: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30s, annealing at 59.2 ℃ for 60s, for 40 cycles; 10min at 98 ℃.
6. The method of claim 4, further comprising a step of performing nucleic acid quantification of the genomic DNA by UV spectrophotometry before the second step.
7. The method according to claim 4, further comprising the step four of: and (3) judging the result, analyzing and processing the detected fluorescence signal by using QuantaSoft1.7.4 software, wherein the microspheres with the amplitude of more than 3500 are positive microspheres, and the microspheres with the amplitude of less than 3500 are negative microspheres.
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