Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a composition and a kit for detecting aspergillus fumigatus, yarrowia pneumospora and cryptococcus neoformans based on QPCR.
The aim and the technical problems of the invention are realized by adopting the following technical proposal.
In one aspect, the invention provides a composition for QPCR-based detection of Aspergillus fumigatus, yersinia and Cryptococcus neoformans, comprising the following components:
primer pair and probe combination for detecting aspergillus fumigatus target gene, and nucleotide sequence of the primer pair and probe combination is shown as SEQ ID NO:1 to 3;
Primer pair and probe combination for detecting yersinia pneumocystis target gene, and nucleotide sequence thereof is shown in SEQ ID NO:4 to 6;
primer pair and probe combination for detecting target gene of cryptococcus neoformans, and nucleotide sequences of the primer pair and probe combination are shown as SEQ ID NO:7 to 9;
The primer pair and probe combination for detecting the human internal control target gene has the nucleotide sequence shown in SEQ ID NO:10 to 12.
Preferably, the probe is labeled ROX, FAM, HEX or CY5 fluorogenic group at the 5 'end and BHQ1 or BHQ2 at the 3' end.
Preferably, SEQ ID NO:3, marking ROX fluorescence generating groups at the 5 'end of the probe, and marking BHQ2 at the 3' end;
SEQ ID NO:6, marking FAM fluorescence generating groups at the 5 'end of the probe and marking BHQ1 at the 3' end;
SEQ ID NO:9, marking HEX fluorescence generating groups at the 5 'end of the probe and marking BHQ1 at the 3' end;
SEQ ID NO:12, and the probe 5 'end is marked with CY5 fluorogenic group and the 3' end is marked with BHQ2.
Preferably, the components of the composition are present in a mixed form.
In another aspect, the invention also provides the use of a composition as described above for the preparation of a kit for QPCR-based detection of aspergillus fumigatus, yarrowia and cryptococcus neoformans.
In yet another aspect, the invention provides a kit for QPCR-based detection of aspergillus fumigatus, pneumosporium yersinia and cryptococcus neoformans, comprising a composition as described above.
Preferably, the probe for detecting the target gene is used at a concentration of 100. Mu.M.+ -.10. Mu.M, the primer for detecting the target gene is used at a concentration of 100. Mu.M.+ -.10. Mu.M, the probe for detecting the internal standard is used at a concentration of 100. Mu.M.+ -.10. Mu.M, and the primer for detecting the internal standard is used at a concentration of 100. Mu.M.+ -.10. Mu.M.
Preferably, the reaction premix, enzyme-free water and template DNA are also included.
Preferably, the reaction premix comprises a buffer solution, a deoxyribonucleoside triphosphate (dUTP/dNTP) mixture, magnesium ions, a hot start polymerase (Taq enzyme) and Uracil DNA Glycosylase (UDG).
The QPCR technology disclosed by the invention is a high-sensitivity and convenient pathogen detection method. A plurality of different probes can be provided in one fluorescent PCR reaction to label different fluorophores, and simultaneously detect a plurality of target genes (aspergillus fumigatus, yarrowia pneumosporosis and cryptococcus neoformans). Fluorescence selection of probes in 4-fold fluorescence PCR: such as FAM/HEX/ROX/Cy5.
The high specificity of QPCR technology is mainly manifested by the semi-preserved replication of PCR amplification that strictly follows the base pairing rules. Semi-reserved replication is one of the most stringent replication modes worldwide. In addition, because of the base complementation principle, the primer in the reaction system can be renatured with the template DNA only when the primer is completely complementary with the target gene, and the primer extension can be performed, the complementation of the primer and the template is the most basic condition for replication, which on the other hand prescribes high specificity of the PCR reaction. The high sensitivity is mainly characterized in that the template DNA is rapidly increased in an exponential manner in the PCR reaction, the early stage of the amplification reaction is rapidly increased in an exponential manner, the late stage of the amplification reaction is in a platform stage, the target sequence can be increased by more than one million times through 45 cycles, and trace target objects can be detected.
The QPCR technology is simple, convenient and quick, the PCR technology can be automatically finished by using Taq enzyme, and a plurality of components are premixed to reduce the sample adding step, so that the amplification effect of PCR is not affected by the simplified step. The detection result of the pathogenic bacteria can be known within 1-2 hours. The time is significantly shorter than the prior art isolation culture and mNGS.
Although Taq enzyme of the present invention lacks 3 'to 5' exonuclease activity, it is unable to correct for incorrect nucleotide incorporation that occurs during the reaction, and there is a degree of mismatched bases in the replicated new DNA strand. The experimental pollution or the operation pollution is easy to generate false positive results. QPCR of multiple target genes may deteriorate the detection results of some targets due to the amount of reagents, mutual interference between primers, competition of targets of different concentrations for PCR reaction reagents, and the like. But can be optimized by redesigning the primers, probes, and changing the amounts of enzymes, magnesium ions, dNTPs, primers, and probes.
According to the invention, a primer probe with stronger specificity is designed according to a conserved region without obvious mutation of yarrowia pneumocystis, cryptococcus neoformans and aspergillus fumigatus, and a proper primer probe group is selected, so that the primers are not mutually interfered while stable and accurate detection is ensured. Meanwhile, a section of RNaseP high-copy human sequence primer probe is set as an internal control. The dUTP/UDG enzyme is introduced into the amplification system to avoid laboratory pollution, and the dUTP is replaced by dUTP in a PCR product under the condition of a certain temperature and time before the PCR reaction, and the dUTP is removed by the UDG enzyme, so that the extension of the DNA polymerase is avoided, and the reamplified capacity is lost. Meanwhile, aspergillus fumigatus, yarrowia pneumospori and cryptococcus neoformans are main pathogenic bacteria for causing fungal infection, and a detection kit is designed aiming at the three bacteria, so that the sensitivity is high and the detection rate is good.
The foregoing description is only an overview of the present invention, and is intended to provide a more thorough understanding of the present invention, and is to be accorded the full scope of the present invention.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purposes and the effects of the present invention easy to understand, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a special premix for qPCR (quantitative polymerase chain reaction) by a probe method suitable for a DNA template, and the special premix is prepared by reacting (Northenan, QN 211). The core component DNA polymerase is a new generation of hot start enzyme which is modified based on an antibody method and improves the affinity of a template through upgrading and reconstruction. The optimal Buffer optimized for the qPCR system is matched, so that the amplification specificity, the detection sensitivity and the amplification linearity of low copy genes are remarkably improved, excellent amplification linearity can be obtained in a wide quantitative range, the target genes are accurately quantified and detected, and the repeatability and the reliability are high. The dUTP/UDG anti-pollution system is added into the reagent, so that the reagent can play a role at room temperature, the influence of amplification product pollution on qPCR can be eliminated, and the accuracy of a result is ensured. The reagent of the reaction system is premixed in advance, the reagent adding operation time is shortened, and the detection time is 1.2 hours.
The reaction premix, unless otherwise specified, includes a buffer, a deoxyribonucleoside triphosphate mixture, magnesium ions, a hot start polymerase, and uracil DNA glycosylase.
Example 1: primer and probe combinations used in the present invention
1. The invention analyzes several virus sequences and designs primer and probe groups shown in table 1 through a great deal of research and exploration:
TABLE 1 primer and probe combinations of the present invention
2. Primer probe combinations Cheng Yeshi pneumospore bacteria, cryptococcus neoformans and aspergillus fumigatus were prepared according to the ratios shown in table 2:
TABLE 2 primer/probe MIX configuration Table for Yersinia, cryptococcus neoformans and Aspergillus fumigatus
3. The invention sets a QPCR experiment system and a program to perform subsequent primer probe tests and experimental operations according to the following tables 3 and 4:
Table 3 reaction system.
Table 4 reaction procedure
4. The yin-yang coincidence rate of the invention accords with the actual sample condition, and the specific result is shown in table 5; the amplification efficiency is 90% -110%; detection limit 20 copies/reaction; the time period from the sample to be detected to the detection result is 2.2h, which is obviously shorter than mNGS.
The specific verification results are as follows:
4.1 yin-yang match rate
The template DNA sample was derived from: selecting 16 cases of positive alveolar lavage fluid samples of clinically definite aspergillus fumigatus or yarrowia pneumospori or cryptococcus neoformans, and numbering the samples in sequence to be 1-16;1 negative alveolar lavage fluid sample, number 17. DNA (Tiangen, DP 316) was extracted from all samples and the resulting nucleic acids were used as templates for fluorescent quantitative detection. Evaluation criteria: the negative-positive coincidence rate is 100 percent.
Conclusion: the Y.yersinia total detection method comprises the steps of detecting 16 samples, wherein 6 positive samples are detected, and the positive coincidence rate is 100%; the cryptococcus neoformans detects 16 samples in total, wherein 5 positive samples are detected, and the positive coincidence rate is 100 percent; the aspergillus fumigatus detects 16 samples in total, wherein 5 positive samples are detected, 5 cases are detected, and the positive coincidence rate is 100%; the negative compliance was 100%. (see Table 5 below)
TABLE 5 comparison of yin-yang compliance of the invention and mNGS
4.2 Comparison of amplification efficiencies
Yarrowia, cryptococcus neoformans and Aspergillus fumigatus plasmid DNA synthesized by Bailigo biotechnology (Shanghai) were selected and diluted in a gradient. Four consecutive gradients were selected for amplification efficiency validation, and each extraction was repeated 2 times. Evaluation criteria: the amplification efficiency is 90% -110%.
Conclusion: the amplification efficiency is in the range of 90% -110%, and the performance meets the requirements. (results are shown in Table 6 below, FIG. 1 to 6)
TABLE 6 detection of CT values for different pathogen concentrations according to the invention
4.3 Detection limit for Yersinia pneumosporosis, aspergillus fumigatus and Cryptococcus neoformans
Yarrowia, cryptococcus neoformans, aspergillus fumigatus plasmid DNA synthesized by Bailey biotechnology (Shanghai) Inc. was diluted to low concentrations and each concentration was tested repeatedly 20 times. The lowest concentration with the detection rate of more than 95% is the detection limit of the corresponding disease source.
Conclusion: the detection limit of the yarrowia pneumocystis, the aspergillus fumigatus and the cryptococcus neoformans is 20 copy per reaction, the detection rate is 100 percent, and the detection limit passes the verification. (see Table 7, see Table 9 below)
Table 7 CT values of 10 copies of three bacteria were measured
Table 8 CT values for 20 copies of three bacteria were measured
Table 9 shows CT values of 50 copies of three bacteria
4.4 Comparison of the time duration of the present invention and the prior art
The time from taking the sample to be detected to obtaining the fungus detection result by adopting the QPCR probe method (the invention) is obviously shorter than mNGS technology. (see Table 10 below)
Table 10 comparison of the present invention and mNGS detection durations
Example 2: comparison of detection Performance of pathogenic microorganisms under different methods
1. Sample selection
10 Known Y.pneumocystis positive samples, number 1-10, 10 known Cryptococcus neoformans positive samples, number 11-20, 10 known Aspergillus fumigatus positive samples, number 21-30, 10 negative samples and number 31-40 are selected. Sample types include alveolar lavage fluid and sputum, and sample loads include high, medium, and low.
2. Nucleic acid extraction:
the DNA of a positive sample of Mycobacterium tuberculosis was extracted using a commercial nucleic acid DNA extraction kit (Tiangen, DP 316) as follows:
10 mL of 10 different tuberculosis drug-resistant positive samples are respectively added into a sterile tube, the centrifugation is carried out at 800 rpm for 5 min, and the supernatant is discarded;
adding 200 mu L of buffer GA to the sediment for resuspension, and transferring all the suspension to a 1.5 mL centrifuge tube;
adding 20 mu L of proteinase K solution, mixing uniformly by vortexing 10 s, standing at 56 ℃ for 60min, and mixing uniformly by vortexing for several times every 15 min;
Adding 200 mu L of buffer solution GB and 1 mu L CARRIER RNA of storage solution, fully reversing and uniformly mixing, placing 10 min at 70 ℃, swirling 10 s every 3 min in the period, clearing the solution at the moment, and centrifuging briefly to remove liquid drops on the inner wall of the pipe cover;
Adding 200 mu L of absolute ethyl alcohol, fully reversing and uniformly mixing, and centrifuging briefly to remove liquid drops on the inner wall of the pipe cover;
Adding the solution obtained in the last step and flocculent precipitate into an adsorption column CR2, centrifuging 12000 rpm for 30 s, and discarding the waste liquid;
adding 500 mu L buffer GD into an adsorption column CR2, centrifuging by 12000 rpm to 30 s, and discarding the waste liquid;
adding 600 mu L of rinsing liquid GD into the adsorption column CR2, centrifuging the solution by 12000 rpm for 30 s, discarding the waste liquid, and repeating the step once again;
12000 Centrifuging at rpm for 2 min, discarding the waste liquid, placing the adsorption column CR2 at room temperature for 2-5 min, and airing the rinse liquid in the adsorption material;
transferring the adsorption column CR2 into a new centrifuge tube, suspending and dropwise adding 25 mu L of elution buffer solution TB to the middle position of the adsorption film, standing for 2-5 min at room temperature, centrifuging at 12000 rpm for 2min min, and collecting the solution into the centrifuge tube to obtain the solution which is the DNA of the sample.
3. In addition, primers and probes of the yersinia pneumococci, the cryptococcus neoformans and the aspergillus fumigatus are designed, and the detection performance of the yersinia pneumoconii, the cryptococcus neoformans and the aspergillus fumigatus is compared by using different primer and probe combinations.
3.1 The primers and probes designed in addition are shown in the following table 11:
table 11 primer probe combinations
3.2 Primer probe combinations Cheng Yeshi pneumospore bacteria, cryptococcus neoformans and aspergillus fumigatus were prepared according to the ratios shown in table 12, and primer/probe MIX-2:
TABLE 12 primer/probe MIX-2 configuration Table for Yersinia, cryptococcus neoformans and Aspergillus fumigatus
3.3 Performing fluorescent PCR detection on the extracted nucleic acid. Subsequent primer probe tests were performed with the following QPCR assay systems and procedures, and experimental procedures, were performed as follows in tables 13 and 14:
TABLE 13 reaction system
Table 14 reaction procedure
3.4 The ct values of the results are recorded in table 18.
4. The method of the invention is used for fluorescence PCR detection of the extracted nucleic acid.
4.1 The mixing of primers and probes was performed according to the following table 15, facilitating the subsequent experimental operations:
TABLE 15 primer/probe MIX configuration Table for Yersinia pneumocandi, cryptococcus neoformans and Aspergillus fumigatus
4.2 And (3) performing fluorescence PCR detection on the extracted nucleic acid. Subsequent primer probe tests, and experimental procedures were performed by setting up QPCR experimental systems and procedures as follows in tables 16 and 17:
TABLE 16 reaction system
Table 17 reaction procedure
4.3 The ct values of the results are recorded in table 18.
5. Results of comparative experiments
By using different fluorescent PCR primers and probes and comparing the detection results of the same nucleic acid, the detection rate of the primers and probes is higher than that of a comparison experiment.
Table 18 comparison of the detection rates of the different methods
Isothermal nucleic acid amplification techniques have evolved rapidly in recent years, being able to be carried out for 20 minutes at constant temperature, and therefore do not require thermal cycling equipment, making them a convenient and rapid tool for pathogen detection. Multi-enzyme isothermal rapid amplification (MIRA) is a modified version of Recombinase Polymerase Amplification (RPA), which is an isothermal nucleic acid amplification technique that can be performed at constant temperature without thermal cycling. With the aid of the helper protein and single-stranded DNA binding protein (SSB), the recombinase and primer form a D-loop region, when the target region is complementary to the primer, the recombinase deflects, and the polymerase binds to the 3' end of the primer and begins chain extension, rapidly completing amplification of the target fragment at 37℃to 42℃for 20 minutes. Compared with LAMP, ELISA and real-time fluorescence quantitative PCR, the MIRA has faster and simpler running speed, only needs a pair of primers, has lower temperature, has the temperature of 37-42 ℃ and has the running time of less than 20 minutes.
RPA technology mainly includes two enzymes: recombinant enzyme T4 UvsX and Bacillus subtilis Pol I. In addition, the RPA reaction system requires template DNA, primers and various materials. The basic reaction process of RPA is as follows: first, the recombinase protein UvsX binds to the RPA primer in the presence of ATP and polyethylene glycol to form a recombinase-primer complex. The complex can then find homologous sequences in the double stranded DNA template. Once a homologous sequence is found, it inserts into the template strand forming a D-ring structure and begins the strand displacement reaction. To prevent the inserted primer from being ejected by branching migration, a substituted template strand is bound to the single-stranded binding protein to maintain stability of the single strand. Finally, the recombinant enzyme is isolated from the complex. In the presence of dNTPs, DNA polymerase binds to the 3' -OH end of the primer to undergo strand elongation to form a new complementary strand. Repeating the steps to realize the exponential amplification of the target area on the template. The overall RPA flow is very fast. Typically, detectable amplification products can be obtained in about 20 minutes. The RPA amplification product may be visualized by conventional agarose gel electrophoresis. Furthermore, the usual RPA methods are mainly exo-RPA and LFS-RPA.
The isothermal rapid amplification technology and the derivative technology are similar to the QPCR technology, and detection needs to be realized by designing primer probes of pathogenic bacteria, except that the isothermal rapid amplification technology can amplify a target fragment only at one temperature, and QPCR is required to amplify the target fragment through temperature change, but the primer probes of yarrowia, cryptococcus neoformans and aspergillus fumigatus are involved, and even the amplification technologies are different, responsibility should be pursued.
While the invention has been described with respect to preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and that any such changes and modifications as described in the above embodiments are intended to be within the scope of the invention.