CN113774157A - Method for rapidly detecting and typing five plasmodia - Google Patents
Method for rapidly detecting and typing five plasmodia Download PDFInfo
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- 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
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Abstract
The invention discloses a set of primer probes for rapidly detecting and parting five plasmodia, which is characterized in that: the method comprises the following steps: a pair of general primers and probes for detecting five plasmodia are respectively NF, NR and NP, two pairs of common primers for distinguishing the five plasmodia are respectively AF, AR, BF and BR, and five hydrolysis probes for distinguishing the five plasmodia are respectively pf-P, pv-P, po-P, pm-P, pk-P, which are designed according to the 18S gene conserved fragments of the pfs, the pvs, the pos, the pm and the pk of the plasmodia, the lengths of the nucleotide sequences amplified by any target gene are within 130 bp; compared with products with amplified fragments exceeding 200bp, the invention reduces the extension time in a mode of reducing the amplified fragments, can make accurate and rapid clinical auxiliary diagnosis on suspected malaria patients in about 64 minutes, and makes typing judgment on 5 plasmodia on positive patients.
Description
The technical field is as follows:
the invention belongs to the technical field of molecular biology detection, and particularly relates to a method for rapidly detecting and typing five plasmodia.
Background art:
for a long time, only four species of plasmodium have been thought to infect humans, namely plasmodium falciparum, plasmodium vivax, plasmodium malariae, plasmodium ovale, and WHO malaria experts have suggested: p. knowlesi has already begun to spread in humans, who are likely to face the threat of infection with the fifth species of Plasmodium. In China, malaria infection mostly occurs in China and south China, mainly including infection of plasmodium falciparum and plasmodium vivax, and malaria is a disease seriously harming human health, so a laboratory detection technology of malaria plays a vital role.
At present, the common detection methods of plasmodium comprise microscopy, immunochromatography, PCR amplification, loop-mediated isothermal amplification and the like. Microscopic examination is a gold standard for detecting plasmodium, but microscopic examination needs to be carried out, and strong specialty is needed for personnel requirements, smear and staining requirements, microscopic examination process and the like; immunological detection such as immunochromatography and the like for plasmodium lactate dehydrogenase has the advantages of simple operation, lower requirements for personnel, improvement on the defect of low microscopic examination repeatability, but the detection accuracy is far lower than that of nucleic acid detection, and the nucleic acid detection technology has important advantages of extremely high accuracy and sensitivity for a part of samples with low number of parasites; the nucleic acid PCR amplification detection method has the advantages of simple design, stable product performance, high detection flux, difficult occurrence of false positive and the like; products for detecting plasmodium on the market are mainly designed around plasmodium falciparum, plasmodium vivax, plasmodium malariae and plasmodium ovale, the plasmodium knowlesi is ignored frequently, and the nucleotide sequence of the plasmodium knowlesi has high similarity with the plasmodium vivax, so that the risk that the plasmodium knowlesi can cause misdetection of the plasmodium vivax is not considered when a primer probe is designed; on the other hand, according to some patents and related documents, the conventional products on the market for detecting plasmodium nucleic acid design universal primers with amplified fragments exceeding 200bp, and when the amplified fragments are too long, the primers need longer time to extend, so that the detection time is increased to some extent.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
The invention content is as follows:
the present invention aims to provide a method for rapidly detecting and typing five plasmodia, thereby overcoming the defects in the prior art.
In order to achieve the above object, the present invention provides a set of primer probes for rapidly detecting and typing five plasmodia, comprising: a pair of general primers and probes for detecting five plasmodia are respectively NF, NR and NP, two pairs of common primers for distinguishing the five plasmodia are respectively AF, AR, BF and BR, and five hydrolysis probes for distinguishing the five plasmodia are respectively pf-P, pv-P, po-P, pm-P, pk-P, which are designed according to the 18S gene conserved fragments of the pfs, the pvs, the pos, the pm and the pk of the plasmodium knowlesi, wherein the amplified nucleotide sequence of any target gene is within 130 bp.
The invention relates to a method for designing a specific primer probe in a 130bp nucleotide sequence by utilizing the technical principle of PCR amplification. Compared with products with amplified fragments exceeding 200bp, the invention reduces the extension time in a mode of reducing the amplified fragments, can make accurate and rapid clinical auxiliary diagnosis on suspected malaria patients in about 64 minutes, and makes typing judgment on 5 plasmodia on positive patients.
Preferably, in the above technical scheme, the NF, NR, NP, AF, AR, pf-P, pv-P are combined into group A reaction solution according to a certain reaction concentration, and the BF, BR, po-P, pm-P, pk-P are combined into group B reaction solution according to a certain reaction concentration.
Preferably, in the technical scheme, the method further comprises the steps of designing a pair of reference gene primers and probes respectively as EF, ER and EP according to the P gene of the human-derived ribonuclease, combining the NF, NR, NP, AF, AR, pf-P, pv-P, EF, ER and EP into a group A reaction solution according to a certain reaction concentration, and combining the BF, BR, po-P, pm-P, pk-P, EF, ER and EP into a group B reaction solution according to a certain reaction concentration.
Preferably, in the above technical scheme, the universal primers and probes NF, NR, NP for detecting five plasmodia are specifically: a universal primer NF: 5'-GAGGAATGCCTAGTAAGCATGATT-3' the flow of the air in the air conditioner,
the general primer NR: 5'-TCCAAACAATTCATCATATCTTTCA-3' the flow of the air in the air conditioner,
the general probe NP is as follows: 5 '-CY 5-TGACTACGTCCCTGCCCTTTGTACAC-BHQ 3-3'.
Preferably, in the above technical scheme, the common primers and specific probes for amplifying plasmodium falciparum and plasmodium vivax:
common primer AF: 5 '-GTGCTACACTGATATRTATAACGAGTTWTT-3',
the common primer AR: 5 '-AGATTACCTACRCYTTTCRGYGG-3',
plasmodium vivax probe pv-P: 5 '-VIC-ATTCAGCTTGCTGTTTCGTATTT-MGB-3',
plasmodium falciparum probe pf-P: 5 '-FAM-TTTGCATACTTTTCCTCCGCCGA-BHQ 1-3'.
Preferably, in the above technical scheme, the sequences of the common primers and the specific probes for amplifying plasmodium malariae, plasmodium ovale and plasmodium knowlesi are as follows:
common primer BF: 5'-CACGCGTGCTACACTGATATG-3' the flow of the air in the air conditioner,
the common primer BR: 5 '-ACGATATGTAYTGATAAAGATTACCTACAC-3',
plasmodium ovale probe po-P: 5 '-VIC-CTCTTTCAAATTGTATAGTTTTCCTC-MGB-3',
plasmodium malariae probe pm-P: 5 '-CY 5-TGCAAAGAATATATAGTTTTCCTCCA-MGB-3',
p. knowlesi probe pk-P: 5 '-FAM-CGATTTTAGCTGCTTGCAGTTTA-BHQ 1-3'.
Preferably, in the above technical scheme, the sequences of primers and probes for amplifying reference genes are as follows:
internal reference gene primer EF: 5'-AGATTTGGACCTGCGAGCG-3' the flow of the air in the air conditioner,
reference gene primer ER: 5'-GAGCGGCTGTCTCCACAAGT-3' the flow of the air in the air conditioner,
reference gene probe EP: 5 '-ROX-TTCTGACCTGAAGGCTCTGCGCG-BHQ 2-3'.
A kit for rapidly detecting and typing five plasmodia, which comprises the primer probe composition in any one of the schemes.
A method for rapidly detecting and typing five plasmodia specifically comprises the following steps:
step 1, extracting template DNA: extracting DNA in a sample to be detected by using a bacterial DNA extraction kit;
step 2.1, 1cycle at 95 ℃ for 10 min;
step 2.2, 95 ℃ X10 sec, 55 ℃ X30 sec, 40 cycles;
in the step of
Collecting fluorescence signals of FAM, VIC, ROX and CY5 channels at 55 ℃ for 30 s;
and 3, interpretation of results:
step 3.1, positive judgment value:
detecting critical positive samples and negative samples, drawing an ROC curve by adopting a statistical method, and determining a positive judgment value by adopting a Youden index;
positive: the Ct of the detection result is less than or equal to 38, the curve is typical S, and the exponential growth period is obvious;
negative: ct > 40 or not detected;
suspected: ct is more than 38 and less than or equal to 40, the suspected sample needs to be rechecked, the Ct value is still in the interval, and the curve is typical S and has obvious index increment period, the sample can be judged to be positive, otherwise, the sample is judged to be negative;
and 3.2, interpreting the result: when the CY5 channel result of the group A is positive, the detection result of the sample can be judged to be positive; when the positive plasmodium is judged, typing the positive plasmodium by referring to the FAM and VIC of the A group and the FAM, VIC and CY5 channel judgment results of the B group; when the signals of the ROX channels of the group A and the group B are positive and the other channels of the two tubes are negative, the detection result of the sample can be judged to be negative; when the CY5 channel result of the group A is negative and the ROX channel signals of the group A and the group B are negative, the detection result of the sample is judged to be unqualified, and resampling and detection are needed.
Compared with the prior art, the invention has the following beneficial effects:
a) the method is convenient and quick, can identify whether the detection sample is a plasmodium positive sample within 64min, and can perform plasmodium typing on the positive sample.
b) The invention designs the common primer according to the 18S gene sequences of five plasmodia, and compared with other conventional PCR products, the product reduces materials in a system and greatly improves the stability of the product.
c) The result of the product is positive and typing, and the interpretation result is more accurate.
d) Has high specificity, and has no cross reaction with other pathogens (toxoplasma gondii, phagocytophilic anaplasma, brucella, neisseria meningitidis, streptococcus, leptospira, salmonella typhi, salmonella paratyphi) which are the same as the infection part or similar to the infection symptoms and are common, and human total nucleic acid.
e) The detection sensitivity is high, and 1000copies/ml plasmodium nucleic acid can be detected at the lowest. The invention has higher auxiliary diagnosis effect on the diagnosis and treatment of common malaria and suspected malaria, and is suitable for popularization.
Description of the drawings:
FIG. 1 is a graph showing the fluorescence PCR amplification curve of positive control and negative control detection.
FIG. 2 is a fluorescence PCR amplification curve diagram of a Plasmodium falciparum positive sample extracted and detected by a nucleic acid extraction kit (SDK60108) of a whole blood sample major organism.
FIG. 3 is a fluorescence PCR amplification graph of Plasmodium vivax positive samples extracted and detected by a nucleic acid extraction kit (SDK60108) of a whole blood sample major.
FIG. 4 is a fluorescence PCR amplification graph of Plasmodium ovale positive samples extracted and detected by a nucleic acid extraction kit (SDK60108) of organisms from a whole blood sample.
FIG. 5 is a fluorescence PCR amplification graph of a Plasmodium malariae positive sample extracted and detected by a nucleic acid extraction kit (SDK60108) of a whole blood sample major organism.
FIG. 6 is a fluorescence PCR amplification graph of a P.knowense positive sample extracted and detected by a nucleic acid extraction kit (SDK60108) from a whole blood sample from a living organism.
FIG. 7 shows the specificity analysis, which is used to extract and detect positive toxoplasma gondii, phagocytophilic anaplasma, Brucella, Neisseria meningitidis, streptococcus, Leptospira, Salmonella typhi, Salmonella paratyphi and human leukocytes thereof by using nucleic acid extraction kit (SDK60108) from major organisms.
FIG. 8 is a schematic diagram showing the design pattern of all primer probes of the present invention.
The specific implementation mode is as follows:
the following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.
Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
The invention designs a pair of universal primer probes (NF, NR and NP) for detecting plasmodium according to 18S gene conserved fragments of plasmodium falciparum (pf), plasmodium vivax (pv), plasmodium ovale (po), plasmodium malariae (pm) and plasmodium knowlesi (pk), two pairs of common primers (AF, AR, BF and BR) for distinguishing five plasmodium parasites and five pairs of hydrolysis probes (pf-P, pv-P, po-P, pm-P, pk-P) for distinguishing five plasmodium parasites; then, designing a pair of reference gene primer probes (EF, ER and EP) according to the P gene of the human-derived ribonuclease in a distinguishing way, finally combining AF, AR, NF, NR, pf-P, pv-P, NP, EF, ER and EP into a group A reaction solution, and combining BF, BR, po-P, pm-P, pk-P, EF, ER and EP into a group B reaction solution. A. And B, adding the two groups of reaction liquid into a sample, amplifying on a fluorescent PCR instrument by using a PCR amplification technology, judging whether plasmodium nucleic acid exists in the sample according to amplification signals of different channels, and carrying out plasmodium typing on the nucleic acid determined as plasmodium.
3.2.1 design patterns of all primer probes of the invention are shown in FIG. 8:
3.2.2: the specific primers and probes required by the invention comprise:
(1) the preferred final reaction concentrations of the common primer probe for amplifying the five plasmodia are respectively 400nM, 400nM and 300nM, and the sequences are as follows:
a universal primer NF: 5'-GAGGAATGCCTAGTAAGCATGATT-3'
The general primer NR: 5'-TCCAAACAATTCATCATATCTTTCA-3'
Universal probe NP (CY 5 fluorescein marker): 5 '-CY 5-TGACTACGTCCCTGCCCTTTGTACAC-BHQ 3-3'
(2) The common primer and the specific probe for amplifying the plasmodium falciparum and the plasmodium vivax preferably have the final reaction concentrations of 500nM, 400nM and 300nM respectively and the sequences as follows:
common primer AF: 5 '-GTGCTACACTGATATRTATAACGAGTTWTT-3'
The common primer AR: 5 '-AGATTACCTACRCYTTTCRGYGG-3'
Plasmodium vivax probe pv-P (VIC fluorescein labeled): 5 '-VIC-ATTCAGCTTGCTGTTTCGTATTT-MGB-3'
Plasmodium falciparum probe pf-P (FAM fluorescein labeled): 5 '-FAM-TTTGCATACTTTTCCTCCGCCGA-BHQ 1-3'
(3) The common primer and the specific probe for amplifying the plasmodium malariae, the plasmodium ovale and the plasmodium knowlesi preferably have the final reaction concentrations of 500nM, 400nM and 400nM respectively, and the sequences are as follows:
common primer BF: 5'-CACGCGTGCTACACTGATATG-3'
The common primer BR: 5 '-ACGATATGTAYTGATAAAGATTACCTACAC-3'
Plasmodium ovale probe po-P (VIC fluorescein labeled): 5 '-VIC-CTCTTTCAAATTGTATAGTTTTCCTC-MGB-3'
Plasmodium malariae probe pm-P (CY 5 fluorescein label): 5 '-CY 5-TGCAAAGAATATATAGTTTTCCTCCA-MGB-3'
P. knowlesi probe pk-P (FAM fluorescein labeled): 5 '-FAM-CGATTTTAGCTGCTTGCAGTTTA-BHQ 1-3'
(4) The primer probes for amplifying the internal reference gene preferably have final reaction concentrations of 300nM (200 nM), 300nM (200 nM) and 200nM (100 nM), respectively, and the sequences are as follows:
internal reference gene primer EF: 5'-AGATTTGGACCTGCGAGCG-3'
Reference gene primer ER: 5'-GAGCGGCTGTCTCCACAAGT-3'
Reference gene probe EP (ROX fluorescein marker): 5 '-ROX-TTCTGACCTGAAGGCTCTGCGCG-BHQ 2-3'
3.2.3: the invention also provides a plasmodium nucleic acid typing detection kit based on real-time fluorescence RT-PCRC, which comprises the primer probe composition.
Further comprises RT-PCR reaction liquid, enzyme mixed liquid, negative control and positive control.
Preferably, the RT-PCR reaction solution comprises buffer solution and Mg2+、dNTPs。
Preferably, the enzyme mixture includes an rnase inhibitor and a DNA polymerase.
Preferably, the positive control is an artificially synthesized gene.
Preferably, the blank is sterilized water with RNase removed. :
3.2.4: a method for rapidly detecting and typing five plasmodia specifically comprises the following steps:
(1) extracting template DNA: the DNA in the sample to be tested was extracted by using the following extraction reagents, a bacterial DNA extraction Kit (cat # SDK60108) from Shuichi, a QIAamp DNA Mini Kit (cat # 51304, 51306) from Qiagen, and nucleic acids were extracted according to the Kit instructions.
(2) Real-time fluorescent PCR amplification reaction: preparing a group A (group B) reaction system in a 100 mul or 200 mul PCR tube: 7.5 muL of primer probe mixed liquid of the group A (group B), 7.5 muL of Taqman OneStep Buffer, 5 muL of enzyme mixed liquid and 5 muL of template DNA, placing the reaction tube in a real-time fluorescence PCR instrument, and setting the following programs for PCR amplification:
step (ii) of
95℃×5min（1cycles）
Step (ii) of
95℃×10sec，55℃×30sec（40cycles）
In the step of
Collecting fluorescence signals of FAM, VIC, ROX and CY5 channels at 55 ℃ for 30 s; the ABI7500 real-time fluorescent PCR instrument selects no ROX correction, and the quenching group selects None. )
The Taqman OneStep Buffer and the enzyme mixed solution are purchased by Shanghai ShuoGui biological company, wherein the product number of the Taqman OneStep Buffer is CSBF-006, and the product number of the enzyme mixed solution is: CSEN-004L.
The real-time fluorescent PCR instrument can select fluorescent quantitative PCR instruments such as ABI7500/7500 fast, QuantStudio 5, Bio-Rad CFX96, Shanghai macrostone SLAN-96P/S, Roche LightCycler480 II and the like.
(3) And (4) analyzing results: ABI7500, ABI Q7, ABI Q5: and automatically storing the result after the reaction is finished, adjusting the Start value, the End value and the Threshold value of Baseline according to the analyzed image (the Start value can be automatically adjusted according to the actual situation, the Start value can be 3-15, the End value can be set at 5-20, the amplification curve of blank contrast is adjusted to be straight or lower than a Threshold line), clicking Analysis to automatically obtain the Analysis result, and viewing the result on a Report interface.
(4) And (4) interpretation of results:
(4.1) Positive judgment value:
according to the invention, critical positive samples and critical negative samples are detected, an ROC curve is drawn by adopting a statistical method, and a positive judgment value is determined by adopting a Youden index (Youden index).
Positive: the Ct of the detection result is less than or equal to 38, the curve is typical S, and the exponential growth period is obvious.
Negative: ct > 40 or not detected.
Suspected: ct is more than 38 and less than or equal to 40, the suspected sample needs to be rechecked, the Ct value is still in the interval, and the curve is typical S and has obvious index increment, the sample can be judged to be positive, otherwise, the sample is judged to be negative.
(4.2) interpreting the result:
when the CY5 channel result of the group A is positive, the detection result of the sample can be judged to be positive; when the positive plasmodium is judged, the positive plasmodium can be classified by referring to the judgment results of the FAM, VIC and B FAM, VIC and CY5 channels.
When the signals of the ROX channels of the group A and the group B are positive and the other channels of the two tubes are negative, the detection result of the sample can be judged to be negative.
When the CY5 channel result of the group A is negative and the ROX channel signals of the group A and the group B are negative, the detection result of the sample is judged to be unqualified, and resampling and detection are needed.
Example 1: designing a primer and a probe:
the method comprises the steps of searching an 18s gene sequence of plasmodium falciparum, an 18s gene sequence of plasmodium vivax, an 18s gene sequence of plasmodium malariae, an 18s gene sequence of plasmodium ovale, an 18s gene sequence of plasmodium knowlesi and a human-derived ribonucleotides P gene sequence through NCBI, comparing the sequences through BioEdit, designing primers and probes through Primer Express 3.0.1, confirming the specificity of the primers and probes through BLAST, and obtaining a universal Primer probe of the plasmodium, two pairs of common primers for distinguishing five plasmodium parasites, five pairs of hydrolysis probes for distinguishing five plasmodium parasites and an internal reference gene Primer probe.
Example 2: extraction of template DNA:
(1) the sample collection comprises a whole blood sample, and the specific collection method comprises the following steps:
it is recommended to collect 2ml of blood sample by using a blood collection tube containing EDTA anticoagulant, and after collection, the blood sample is inverted and mixed uniformly, so that the anticoagulant on the inner wall of the blood collection tube is fully mixed with the blood.
(2) The collected whole blood sample is extracted according to the instruction of the nucleic acid extraction kit, and the following extraction kit can be used: bacterial DNA extraction Kit (cat # SDK60108) from major and QIAamp DNA Mini Kit (cat # 51304, 51306) from Qiagen.
Example 3: preparing a primer probe reaction solution and a kit reaction solution:
(1) preparing a group A primer probe reaction solution:
(2) preparing a B group primer probe reaction solution:
(3) preparing a reaction solution:
example 4: and (3) amplification procedure:
（1）95℃×5min；1cycles，
（2）95℃×10sec，55℃×30sec——40cycles，
collecting fluorescence signals of FAM, VIC, ROX and CY5 channels at the temperature of 55 ℃ in the step (2) and for 30 s; the ABI7500 real-time fluorescent PCR instrument selects no ROX correction, and the quenching group selects None.
Example 5: and (4) interpretation of results:
on the basis of positive control detection, negative control detection and positive sample detection results of ROX of tube A and ROX channel Rnasep of tube B, result interpretation is carried out according to the following table:
a pipe system:
a pipe system:
example 6:
the results are shown in Table 1, and the fluorescence PCR amplification curve is shown in FIG. 7.
Table 1: and (3) specific analysis:
on the basis of the confirmation of the lowest detection limit of plasmodium falciparum, plasmodium vivax, plasmodium malariae, plasmodium ovale and plasmodium knowlesi detected by fluorescence PCR, nucleic acid of plasmodium falciparum, plasmodium vivax, plasmodium malariae, plasmodium ovale and plasmodium knowlesi are respectively extracted by a nucleic acid extraction kit (SDK60108) of major organisms, then the extracted nucleic acid is absolutely quantified by a digital PCR technology, and the nucleic acid with determined concentration is diluted to 10000copies, 1000copies and 100copies for detection, wherein each gradient is repeated for 10 times. The results are shown in Table 2.
Table 2: and (3) confirming the lowest detection limit:
the foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (9)
1. A set of primer probes for rapidly detecting and parting five plasmodia, which is characterized in that: the method comprises the following steps: a pair of general primers and probes for detecting five plasmodia are respectively NF, NR and NP, two pairs of common primers for distinguishing the five plasmodia are respectively AF, AR, BF and BR, and five hydrolysis probes for distinguishing the five plasmodia are respectively pf-P, pv-P, po-P, pm-P, pk-P, which are designed according to the 18S gene conserved fragments of the pfs, the pvs, the pos, the pm and the pk of the plasmodium knowlesi, wherein the amplified nucleotide sequence of any target gene is within 130 bp.
2. The primer probe for rapidly detecting and typing five plasmodia according to claim 1, wherein: the NF, NR, NP, AF, AR and pf-P, pv-P are combined into A group reaction liquid according to certain reaction concentration, and the BF, BR and po-P, pm-P, pk-P are combined into B group reaction liquid according to certain reaction concentration.
3. The primer probe for rapidly detecting and typing five plasmodia according to claim 2, wherein: the kit also comprises a pair of reference gene primers and probes which are respectively EF, ER and EP and are designed according to the gene P of the humanized ribonuclease, wherein the NF, NR, NP, AF, AR, pf-P, pv-P, EF, ER and EP are combined into a group A reaction solution according to certain reaction concentration, and the BF, BR, po-P, pm-P, pk-P, EF, ER and EP are combined into a group B reaction solution according to certain reaction concentration.
4. The primer probe for rapidly detecting and typing five plasmodia according to claim 1, wherein: the general primers and probes NF, NR and NP for detecting five plasmodia are specifically as follows:
a universal primer NF: 5'-GAGGAATGCCTAGTAAGCATGATT-3' the flow of the air in the air conditioner,
the general primer NR: 5'-TCCAAACAATTCATCATATCTTTCA-3' the flow of the air in the air conditioner,
the general probe NP is as follows: 5 '-CY 5-TGACTACGTCCCTGCCCTTTGTACAC-BHQ 3-3'.
5. The primer probe for rapidly detecting and typing five plasmodia according to claim 1, wherein: a common primer and a specific probe for amplifying plasmodium falciparum and plasmodium vivax:
common primer AF: 5 '-GTGCTACACTGATATRTATAACGAGTTWTT-3',
the common primer AR: 5 '-AGATTACCTACRCYTTTCRGYGG-3',
plasmodium vivax probe pv-P: 5 '-VIC-ATTCAGCTTGCTGTTTCGTATTT-MGB-3',
plasmodium falciparum probe pf-P: 5 '-FAM-TTTGCATACTTTTCCTCCGCCGA-BHQ 1-3'.
6. The primer probe for rapidly detecting and typing five plasmodia according to claim 1, wherein: the common primer and the specific probe for amplifying the plasmodium malariae, the plasmodium ovale and the plasmodium knowlesi have the following sequences:
common primer BF: 5'-CACGCGTGCTACACTGATATG-3' the flow of the air in the air conditioner,
the common primer BR: 5 '-ACGATATGTAYTGATAAAGATTACCTACAC-3',
plasmodium ovale probe po-P: 5 '-VIC-CTCTTTCAAATTGTATAGTTTTCCTC-MGB-3',
plasmodium malariae probe pm-P: 5 '-CY 5-TGCAAAGAATATATAGTTTTCCTCCA-MGB-3',
p. knowlesi probe pk-P: 5 '-FAM-CGATTTTAGCTGCTTGCAGTTTA-BHQ 1-3'.
7. The primer probe for rapidly detecting and typing five plasmodia according to claim 1, wherein: the sequences of primers and probes for amplifying the reference gene are as follows:
internal reference gene primer EF: 5'-AGATTTGGACCTGCGAGCG-3' the flow of the air in the air conditioner,
reference gene primer ER: 5'-GAGCGGCTGTCTCCACAAGT-3' the flow of the air in the air conditioner,
reference gene probe EP: 5 '-ROX-TTCTGACCTGAAGGCTCTGCGCG-BHQ 2-3'.
8. A detection kit for rapidly detecting and typing five plasmodia is characterized in that: comprising a primer probe composition according to any one of claims 1 to 7.
9. A method for rapidly detecting and typing five plasmodia specifically comprises the following steps:
step 1, extracting template DNA: extracting DNA in a sample to be detected by using a bacterial DNA extraction kit;
step 2, real-time fluorescence PCR amplification reaction: respectively preparing a group A reaction system and a group B reaction system in 100 mul or 200 mul PCR tubes: 7.5 muL of primer probe mixed liquid of the group A and the group B, 7.5 muL of Taqman OneStep Buffer, 5 muL of enzyme mixed liquid and 5 muL of template DNA, placing the reaction tube in a real-time fluorescence PCR instrument, and setting the following programs for PCR amplification:
step 2.1, 1cycle at 95 ℃ for 5 min;
step 2.2, 95 ℃ X10 sec, 55 ℃ X30 sec, 40 cycles;
in the step of
Collecting fluorescence signals of FAM, VIC, ROX and CY5 channels at 55 ℃ for 30 s;
and 3, interpretation of results:
step 3.1, positive judgment value:
detecting critical positive samples and negative samples, drawing an ROC curve by adopting a statistical method, and determining a positive judgment value by adopting a Youden index;
positive: the Ct of the detection result is less than or equal to 38, the curve is typical S, and the exponential growth period is obvious;
negative: ct > 40 or not detected;
suspected: ct is more than 38 and less than or equal to 40, the suspected sample needs to be rechecked, the Ct value is still in the interval, and the curve is typical S and has obvious index increment period, the sample can be judged to be positive, otherwise, the sample is judged to be negative;
and 3.2, interpreting the result: when the CY5 channel result of the group A is positive, the detection result of the sample can be judged to be positive; when the positive plasmodium is judged, typing the positive plasmodium by referring to the FAM and VIC of the A group and the FAM, VIC and CY5 channel judgment results of the B group; when the signals of the ROX channels of the group A and the group B are positive and the other channels of the two tubes are negative, the detection result of the sample can be judged to be negative; when the CY5 channel result of the group A is negative and the ROX channel signals of the group A and the group B are negative, the detection result of the sample is judged to be unqualified, and resampling and detection are needed.
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