CN115820891A - Primer probe combination, kit and detection method for detecting and parting 7 plasmodia - Google Patents
Primer probe combination, kit and detection method for detecting and parting 7 plasmodia Download PDFInfo
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Abstract
The invention provides a primer probe combination, a kit and a detection method for detecting and parting 7 plasmodium, wherein the 7 plasmodium are plasmodium falciparum pf-a in asexual stage, plasmodium falciparum pf-s in sexual stage, plasmodium vivax pv vivax, plasmodium ovale poc subtype, plasmodium ovale pow subtype, plasmodium malariae pm and plasmodium knowlesi pk, and the primer probe combination comprises: fp1, rp1, tp1; rp2; tp2, tp3, tp4, tp5, tp6, tp7, tp8; fp2, fp3, fp4, fp5, fp6, fp7; fp8, fp9, rp3, tp9. The invention adopts the fluorescence PCR technology combined with melting curve analysis, realizes the high-flux detection of plasmodium breeding types on the basis of PCR high sensitivity and high specificity detection, has ingenious design in the whole process and simple operation, and provides a more convenient detection method for clinic.
Description
Technical Field
The invention relates to a material for analyzing materials by means of chemical or physical properties of the materials, belongs to the field of in-vitro diagnostic reagents, and particularly relates to a primer probe combination and a kit for detecting and parting 7 plasmodia, and a method for detecting human plasmodia by utilizing a fluorescence PCR combined melting curve analysis technology.
Background
Malaria is a life-threatening disease caused by plasmodium. Malaria seriously harms human health, and is listed as a global public health problem needing urgent control by the world health organization together with AIDS and tuberculosis. In 2019 there were 2.41 million cases of malaria, 62.7 million people die, and more than half of the world population faces malaria risk. The pathogenic microorganism plasmodium has complex species, and can easily develop severe malaria with high mortality rate once treatment is delayed, accurate early diagnosis can reduce diseases and prevent death, and is also helpful for reducing malaria transmission.
There are 5 species of human malaria parasites, i.e., plasmodium falciparum (pf), plasmodium vivax (pv), plasmodium ovale (po), plasmodium malariae (pm), plasmodium falciparum (pk), and Plasmodium knowlesi (pk), of which pf and pv are the most harmful. Statistically, pf accounts for 99.7% of the estimated number of malaria cases in the african region of the world health organization in 2017, and most of malaria cases in the southeast asia region (62.8%), the east china sea region (69%), and the western pacific (71.9%) of the world health organization, wherein pf is divided into sexual stage pf-s and asexual stage pf-a according to its life history; pv is the major parasite in the american region of the world health organization, accounting for 74.1% of malaria cases; po is mostly distributed in africa, and in recent years, some scholars classify po into two subtypes, namely poc (plasma over clinical disease) and pow (plasma over Walikeri), and research shows that pow causes more severe thrombocytopenia clinically than poc (p = 0.031), and in addition, the window period of pow is shorter; pm and pk are the two most difficult types to identify clinically, with pm being the species of plasmodium found early to infect humans and pk being the fifth human plasmodium found in recent years.
According to [ WS 259-2015 ] malaria diagnosis, the detection methods for plasmodium mainly include microscopic blood smear, antigen detection and nucleic acid detection. Microscope microscopy is used as a WHO recommended standard detection method, the detection result is reliable, but operators need strict experience requirements, and poc, pow, pm and pk are difficult to distinguish during microscopy, so that the detection requirement is difficult to meet; the antigen detection can realize rapid detection, is one of the detection methods commonly used for pathogenic microorganisms in the current medical detection, but has poor detection sensitivity, and is easy to miss detection for some malaria patients in an infection window period.
The plasmodium has high similarity on genomes, wherein the mutation sites of pf-a and pf-s, poc and pow are few, the misjudgment risk can occur when a real-time fluorescence PCR method is adopted for distinguishing, and the macroscopic difference of Tm values can be reflected by the difference of a melting curve aiming at 1 base, so that the pf-a, pf-s, poc and pow can be accurately judged. There are many methods for realizing the melting curve, most of them adopt asymmetric amplification to realize the melting curve analysis, and the premise of the asymmetric melting curve analysis is that one primer is limited, so that the detection sensitivity is reduced, and secondly, some scholars adopt the ligase technology to realize the melting curve, but the method needs to open the cover after re-amplification, so that the possibility of environmental pollution is increased.
In conclusion, the development of a reagent for identifying the type of malaria parasites, which has a high flux and excellent detection accuracy, is of great importance in the monitoring and clinical diagnosis of malaria.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention combines the melting curve analysis based on the fluorescence PCR technology, realizes the detection of a plurality of genes in a single-color fluorescence channel on the basis of the advantages of rapid and stable PCR, strong specificity, high sensitivity and the like, thereby greatly improving the flux, realizing the qualitative detection of plasmodium in 1 tube of reaction liquid and simultaneously carrying out specific typing analysis on positive plasmodium samples.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the first purpose of the invention is to provide a primer probe combination for detecting and parting 7 plasmodium, wherein the 7 plasmodium is plasmodium falciparum pf-a in asexual stage, plasmodium falciparum pf-s in sexual stage, plasmodium vivax pv, plasmodium ovale poc subtype, plasmodium ovale pow subtype, plasmodium malariae pm, plasmodium knowlesi pk;
the primer probe combination comprises:
universal primers and probes fp1, rp1 and tp1 for detecting plasmodium in human body;
fp1 and rp1 are two plasmodium specific primers used for amplifying infectious human plasmodium, and the Tm value is set to be 60-61 ℃;
tp1 is a specific probe for detecting plasmodium which can infect human, 5 'and 3' respectively mark ROX fluorescein and MGB fluorescence quenching groups, and the Tm value is set at 75-76 ℃.
A primer rp2 for distinguishing the plasmodium types of the human bodies;
rp2 is a specific primer for distinguishing plasmodium species, and the Tm value is set at 60-61 ℃.
Probes tp2, tp3, tp4, tp5, tp6, tp7 and tp8 for distinguishing plasmodium types of human bodies;
tp2, tp3, tp4, tp5, tp6, tp7 and tp8 are plasmodium probes for distinguishing infected persons, pf-a, pf-s, pv, poc, pow, pm and pk are sequentially used for detecting plasmodium types, FAM fluorescein and MGB fluorescence quenching groups are respectively marked on 5 'and 3' of tp2, tp3 and tp4, VIC fluorescein and MGB fluorescence quenching groups are respectively marked on 5 'and 3' of tp5, tp6, tp7 and tp8, and Tm values of the 6 probes are set at 65-75 ℃.
Melting curve primers fp2, fp3, fp4, fp5, fp6, fp7 and fp8 for distinguishing plasmodium types of human bodies;
fp2, fp3, fp4, fp5, fp6, fp7 and fp8 are melting curve primers for distinguishing plasmodium capable of infecting human, nucleotide sequences of 3' ends of the 6 primers are consistent, are specifically complementary with a template, and are used for matching with rp2 to carry out a PCR process, so that the detection effects on tp2, tp3, tp4, tp5, tp6, tp7 and fp8 are achieved; the nucleotide sequence at the 5' end and tp2, tp3, tp4, tp5, tp6, tp7 and fp8 are in a reverse incomplete complementary state, a double-chain structure can be formed at low temperature by a corresponding primer and a probe, a Tm value is generated by melting curve analysis, so that a melting curve graph showing a peak shape is drawn by a fluorescent PCR instrument, each peak in each channel is dispersed and shown by optimizing the sequence, and the peak can represent a specific plasmodium type.
Internal reference gene primers and probes fp9, rp3 and tp9 for monitoring the reaction process;
fp9 and rp3 are two specific primers for the human ribonuclease P, the Tm value is set at 60-61 ℃, tp9 is a specific probe for detecting the human ribonuclease P, 5 'and 3' of the probe are respectively marked with CY5 fluorescein and BHQ3 fluorescence quenching group, and the Tm value is set at 75-76 ℃.
The fp1 nucleotide sequence is shown as SEQ ID NO. 1: 5' GTATTCGCGCGCAAGCGAGAAA-;
the rp1 nucleotide sequence is shown as SEQ ID NO. 2: 5' ACCATCCAAGAAATCAAGAAAGAGC-;
the tp1 nucleotide sequence is shown as SEQ ID NO.3, the 5 'mark ROX fluorescein and the 3' mark MGB fluorescence quenching group of the tp1 are as follows: 5'-ROX-CAGGCGTGGAGCTTGCG-MGB-3';
the rp2 nucleotide sequence is shown as SEQ ID NO. 4: 5 'CCCAGAAACCCAAAAGACTTTGATTT-doped 3';
the tp2 nucleotide sequence is shown as SEQ ID NO.5, the 5' label of tp2 is FAM fluorescein, and the 3' label of 3' is MGB fluorescence quenching group: 5 '-FAM-caacattccctaagaaaatgc-MGB-3';
the nucleotide sequence of tp3 is shown as SEQ ID NO.6, the 5' label of tp3 is FAM fluorescein, and the 3' label of 3' is MGB fluorescence quenching group: 5 '-FAM-TCTAAAAGTCACCTCGAAAAGATG-MGB-3';
the tp4 nucleotide sequence is shown as SEQ ID NO.7, the 5 'label FAM fluorescein and the 3' label MGB fluorescence quenching group of tp4 are as follows: 5 '-FAM-CTCTCTTCGGAGTTTATTCTTAGATTGCTTCC-MGB-3';
the tp5 nucleotide sequence is shown as SEQ ID NO.8, the 5 'mark VIC fluorescein and the 3' mark MGB fluorescence quenching group of tp5 are as follows: 5'-VIC-CCTTTCGG + G + GAAATTTC-MGB-3';
the tp6 nucleotide sequence is shown as SEQ ID NO.9, the 5 'mark VIC fluorescein and the 3' mark MGB fluorescence quenching group of the tp6 are as follows: 5'-VIC-CCTTT + T + G + G + AAATTTCTTAG-MGB-3';
the tp7 nucleotide sequence is shown as SEQ ID NO.10, the 5 'mark VIC fluorescein and the 3' mark MGB fluorescence quenching group of the tp7 are as follows: 5'-VIC-CAC + TCATATA + TA + AGAATGTCTC-MGB-3';
the tp8 nucleotide sequence is shown as SEQ ID NO.11, 5 'mark VIC fluorescein of tp7, 3' mark MGB fluorescence quenching group: 5'-VIC-CTCCGGAGATTAGAACTCTTAG-MGB-3';
the fp2 nucleotide sequence is shown in SEQ ID NO. 12: 5 'GCATTTCTTAGGGAATGTTGGGGAGTGAAGGACGATCAGATACCG-3';
the fp3 nucleotide sequence is shown as SEQ ID NO. 13: 5 'CATATTTAGAGGTGAATTTTAGAGGGGAGTGAAGACGATACCG-3';
the fp4 nucleotide sequence is shown as SEQ ID NO. 14: 5 'GGAAGCAATCTAAGAATAAACTCCGAAGAGGGGAGTGAAGACGATCAGATACCG-3';
the fp5 nucleotide sequence is shown in SEQ ID NO. 15: 5 'GAAATTTCCCCGAAAGGGGAGTGAAGGACGATCAGATACCG 3';
the fp6 nucleotide sequence is shown in SEQ ID NO. 16: 5 'CTAAGAAATTTCCAAAAGGGGGAGTGAAGGAACGAACGATCAGATACCG-3';
the fp7 nucleotide sequence is shown in SEQ ID NO. 17: 5 'GAGAGACATTCTATATATGAGTGGGGAGTGAAGACGATCAGATACCG-3';
the fp8 nucleotide sequence is shown in SEQ ID NO. 18: 5 'CTAAGAGTTCTAATCTCGGAGGGGAGTGAAGACGATCAGATACCG 3';
the fp9 nucleotide sequence is shown in SEQ ID NO. 19: 5 'AGATTTGGACCTGCGAGCG-doped 3';
the rp3 nucleotide sequence is shown as SEQ ID NO. 20: 5 'GAGCGGCTGTCTCCACAAGTT-doped 3';
the tp9 nucleotide sequence is shown as SEQ ID NO.21, the 5 'of tp9 is marked with CY5 fluorescein, and the 3' is marked with BHQ3 fluorescence quenching group: 5'-CY 5-TTCTGACCTGAAGGCTCTGTCGCG-BHQ 3-3'.
The "+" in the nucleotide sequence of the primer of the invention represents the modification of the base-locked nucleic acid, and the modified base is the adjacent base at the 3' end of the "+" nucleotide.
The second purpose of the invention is to provide a kit for detecting and typing 7 plasmodium, wherein the kit comprises the primer and probe combination.
Further, the final concentration of the primer probe reaction in the kit is fp1:400nm, rp1 400nm, tp1.
The final concentration of the primer probe reaction in the invention refers to: when the reagent is prepared and then the upper machine is operated, the concentration of the primer probe in the PCR tube, for example, when the final volume of the detection system is 30. Mu.L or 50. Mu.L, is the concentration of the primer probe.
Further, the kit also comprises a nucleic acid amplification reaction solution, a positive standard, a blank reference substance and a TE buffer solution.
Further, the nucleic acid amplification reaction solution contains one or a mixture of more of tris, potassium chloride, magnesium chloride, ammonium sulfate, dNTP, and Taq DNA polymerase.
Further, the nucleic acid amplification reaction solution contains potassium chloride: 180mM, magnesium chloride: 45mM, ammonium sulfate: 80mM, dATP:20mM, dTTP:20mM, dGTP:20mM, dCTP:20mM, EDTA:2mM, taq DNA polymerase: 40U, pH adjusted to 8.0 using Tris-HCl.
In a specific example, the nucleic acid amplification reaction solution is used in a volume of 12.5. Mu.L for a single aliquot.
Further, the positive standard includes:
a sexual stage plasmodium falciparum pf-s gene fragment with a nucleotide sequence shown as SEQ ID NO. 22:
AAGGGAGTGAAGACGATCAGATACCGTCGTAATCTTAACCATAAACTATACCGACTAGGTGTTGGATGAATATAAAAAATATATAAATATGTAGCATTTCTTAGGGAATGTTGATTTTATATTAGAATTGCTTCCTTCAGTACCTTATGAGAAATCAAAGTCTTTGGGTTCTGGGGCGAGTATTCGCGCAAGCGAGAAAGTTAAAAGAATTGACGGAAGGGCACCACCAGGCGTGGAGCTTGCGGCTTAATTTGACTCAACACGGGAAAACTCACTAGTTTAAGACAAGAGTAGGATTGACAGATTAATAGCTCTTTCTTGATTTCTTGGATGGTGA；
asexual stage plasmodium falciparum pf-a gene fragment with a nucleotide sequence shown as SEQ ID No. 23:
AAGGGAGTGAAGACGATCAGATACCGTCGTAATCTTAACCATAAACTATGCCGACTAGGTGTTGGATGAAAGTGTTAAAAATAAAAGTCATCTTTCGAGGTGACTTTTAGATTGCTTCCTTCAGTACCTTATGAGAAATCAAAGTCTTTGGGTTCTGGGGCGAGTATTCGCGCAAGCGAGAAAGTTAAAAGAATTGACGGAAGGGCACCACCAGGCGTGGAGCTTGCGGCTTAATTTGACTCAACACGGGGAAACTCACTAGTTTAAGACAAGAGTAGGATTGACAGATTAATAGCTCTTTCTTGATTTCTTGGATGGTGA；
a plasmodium vivax pv gene fragment having a nucleotide sequence shown in SEQ ID No. 24:
AAGGGAGTGAAGACGATCAGATACCGTCGTAATCTTAACCATAAACTATGCCGACTAGGCTTTGGATGAAAGATTTTAAAATAAGAATTTTCTCTTCGGAGTTTATTCTTAGATTGCTTCCTTCAGTGCCTTATGAGAAATCAAAGTCTTTGGGTTCTGGGGCGAGTATTCGCGCAAGCGAGAAAGTTAAAAGAATTCGGAAGGGCACCACCAGGCGTGGAGCTTGCGGCTTAATTTGACTCAACACGGGAAAACTCACTAGTTTAAGACAAGAGTAGGATTGACAGATTAATAGCTCTTTCTTGATTTCTTGGATGGTGA；
a plasmodium ovale poc gene fragment having a nucleotide sequence shown in SEQ ID No. 25:
AAGGGAGTGAAGACGATCAGATACCGTCGTAATCTTAACCATAAACTATACCAACTAGGTTTTGGATGAAAAGTTTTTAAATAAGAGAATTCCTTTCGGGGAAATTTCTTAGATTGCTTCTTTCAGTACCTTATGAGAAATCAAAGTCTTTGGGTTCTGGGGCGAGTATTCGCGCAAGCGAGAAAGTTAAAAGAATTGACGGAAGGGCACCACCAGGCGTGGAGCTTGCGCTTAATTTGACTCAACACGGGGAAACTCACTAGTTTAAGACAAGAGTAGGATTGACAGATTAATAGCTCTTTCTTGATTTCTTGGATGGTGA；
plasmodium ovale pow gene fragment with the nucleotide sequence shown as SEQ ID NO. 26:
AAGGGAGTGAAGACGATCAGATACCGTCGTAATCTTAACCATAAACTATGCCGACTAGGTTTTGGATGAAAGATTTTTAAATAAGAAAATTCCTTTTGGAAATTTCTTAGATTGCTTCCTTCAGTACCTTATGAGAAATCAAAGTCTTTGGGTTCTGGGGCGAGTATTCGCGCAAGCGAGAAAGTTAAAAGAATTGACGGAAGGGCACCACCAGGCGTGGAGCTTGCGCTTAATTTGACTCAACACGGGGAAACTCACTAGTTTAAGACAAGAGTAGGATTGACAGATTAATAGCTCTTTCTTGATTTCTTGGATGGTGA；
plasmodium malariae pm gene segment with a nucleotide sequence shown as SEQ ID NO. 27:
AAGGGAGTGAAGACGATCAGATACCGTCGTAATCTTAACCATAAACTATGCCGACTAGGTGTTGGATGATAGTGTAAAAAATAAAAGAGACATTCTTATATATGAGTGTTTCTTTTAGATAGCTTCCTTCAGTACCTTATGAGAAATCAAAGTCTTTGGGTTCTGGGGCGTATTCGCGCAAGCGAGAAAGTTAAAAGAACCGACGGAAGGGGACACAGGCGTGGAGCTTGCGGCTTAATTTGACTCAACACGGGGAAACTCACTAGTTTAAGACAAGAGTAGGATTGACAGATTAATAGCTCTTTCTTGATTTCTTGGATGGTGA；
the nucleotide sequence of the plasmodium knowlesi pk gene segment is shown as SEQ ID NO. 28:
AAGGGAGTGAAGACGATCAGATACCGTCGTAATCTTAACCATAAACTATGCCGACTAGGCTTTGGATGAAAGATTTTAAAATAAGAGTTTTTCTTTTCTCTCCGGAGATTAGAACTCTTAGATTGCTTCCTTCAGTGCCTTATGAGAAATCAAAGTCTTTGGGTTCTGGGGCGAGTATTCGCGCAAGCGAGAAAGTTAAAAGAATTGACGGAAGGGCACCACCAGGCGTGGAGCTTGCGGCTTAATTTGACTCAACACGGGAAAACTCACTAGTTTAAGACAAGAGTAGGATTGACAGATTAATAGCTCTTTCTTGATTTCTTGGATGGTGA；
a ribonucleotide enzyme Rnasep gene fragment with the nucleotide sequence shown as SEQ ID NO. 29:
ATGGGACTTCAGCATGGCGGTGTTTGCAGATTTGGACCTGCGAGCGGGTTCTGACCTGAAGGCTCTGCGCGGACTTGTGGAGACAGCCGCTCACCTTGGCTATTCAGTTGTTGCTATCAATCATATCGTTGACTTTAAGGAAAAGAAA。
further, the positive standard comprises a positive control 1, a positive control 2, a positive control 3 and a positive control 4; the preparation method specifically comprises the following steps:
positive control 1: the concentration ratio of the pf-a gene fragment to the poc gene fragment =10 6 copies/ml：10 6 Preparing copies/ml, and using TE buffer solution as solvent;
positive control 2: pf-s Gene fragment-pow Gene fragment = 2X 10 in concentration ratio 5 copies/ml：5×10 5 Preparing copies/ml, and using TE buffer solution as solvent;
positive control 3: pm Gene fragment =10 as concentration ratio of pv Gene fragment to pm Gene fragment 6 copies/ml：8×10 5 Preparing copies/ml, and using TE buffer solution as solvent;
positive control 4: according to the concentration ratio, the pk gene fragment to the Rnasep gene fragment =10 6 copies/ml：10 7 copies/ml, and the solvent is TE buffer.
Further, the blank control is water sterilized to remove DNA.
The third purpose of the invention is to provide the application of the primer probe combination in preparing the reagent for detecting and typing 7 plasmodium.
It is a fourth object of the present invention to provide a method for detecting and typing 7 plasmodia, the method comprising the steps of:
step 1-1: calculating a preparation amount n, wherein n = detection person +4 positive standard substance + 1 blank reference substance.
In a particular embodiment: for a certain experiment, 20 samples need to be detected, and the preparation amount n =20+4+1=25.
Step 1-2: preparing a plasmodium melting curve detection solution: adding the primer probe combination of claim 1 into a nucleic acid amplification reaction solution, adjusting the volume by using a TE buffer solution to prepare a plasmodium melting curve detection solution, and detecting the plasmodium melting curve
The preparation parts of the test solution are not less than n;
in a particular embodiment, the plasmodium melt curve test solution formulation is performed according to the following table:
extracting DNA from blood of an organism to be detected as template DNA;
the sample DNA extracted in step 2, positive standard, and blank control are added to the split plasmodium melt curve assay solution described in step 1 in 10 μ L volumes, respectively, so that the final concentration of the reaction of the aforementioned primer-probe combination is fp1:400nm, rp1 400nm, tp1; oscillating and centrifuging;
and 4, program operation:
and (3) operating the system obtained in the step (3) on a real-time fluorescent PCR instrument, wherein the operating program is as follows:
step (1): multiplying by 10min at 95 ℃;1cycles
Step (2): 95 ℃ X10 s,58 ℃ X30s, 60 ℃ X10 s;40cycles
And (3): 95 ℃ multiplied by 10s,20-65 ℃ multiplied by 10s, 37 ℃;1cycles
Step (2) and step (3) of collecting fluorescence by setting FAM, VIC, ROX and CY5 at 58 ℃ for 30s and 20-65 ℃; ROX correction is not selected in real-time fluorescent PCR, and None is selected as a quenching group;
step (3) and the temperature of 20-65 ℃ is the temperature rise process of the melting curve, and the temperature rise rate is 0.03 ℃/s.
And 5, analyzing results:
firstly, analyzing a real-time fluorescent PCR amplification result, and directly judging that a sample to be detected is negative if FAM, VIC and ROX channel amplification results are negative and CY5 channel amplification is positive;
if the ROX channel amplification result is positive, the sample to be detected is plasmodium positive, at least one channel amplification result of the FAM and VIC channels is also positive, then melting curve analysis is carried out in the fluorescence channel with the amplification result of the FAM and VIC channels being positive, the specific type of the plasmodium is judged, and the type of the plasmodium corresponding to the disappeared peak is the positive type;
6, judging the real-time fluorescent PCR amplification result in the step 5:
positive judgment value:
judging that the amplification result is positive when the Ct of the detection result is less than or equal to 38 and the melting curve map is obviously different from the blank reference substance;
judging that the amplification result is negative when the detection result Ct is more than 38 or is not detected;
and (3) judging the positive type:
the target genes of FAM channel detection are pf-a, pf-s and pv, when a melting curve is analyzed, three specific main peaks which sequentially appear in a temperature increasing mode respectively represent pf-a, pf-s and pv, and the disappeared peaks are positive types;
the target genes of VIC channel detection are pm, poc, pow and pk, four specific main peaks which sequentially appear in a temperature increasing mode during melting curve analysis respectively represent pm, poc, pow and pk, and the disappeared peaks are positive types;
the target gene of the ROX channel detection is plasmodium universal gene which can infect human,
the target gene of CY5 channel detection is Rnasep; the Rnasep is used as a human-derived internal standard gene and is used for monitoring whether a negative result is a real negative result or a false negative result caused by sample collection failure or extraction failure.
The FAM and VIC channel detection needs to be combined with melting curve analysis and interpretation, the ROX and CY5 channels do not carry out melting curve analysis, and the negative and positive plasmodium of a sample to be detected is interpreted only through a real-time fluorescence PCR amplification result.
Positive standards were used to determine whether the above tests were qualified: when all the sample detection results are negative and the positive standard detection result is positive in the detection result of a certain example, the detection system is qualified, and the sample is negative; and when the detection result of the sample and the detection result of the positive standard substance are negative, the detection result is unreliable or fails, and the re-detection is needed.
The technical principle of the invention is as follows: when a target gene fragment does not exist in a reaction system, the primer probe is in a free state in the system, PCR reaction does not occur in the amplification stage of the reaction program, any primer probe is not consumed in the amplification stage, during melting curve analysis, the fluorescent probe is combined with the 5' end of the corresponding primer to form a double-stranded structure, the double-stranded structure is dissociated in the temperature rising process, and a specific melting curve peak is drawn at the position of the corresponding Tm value by a fluorescent PCR instrument; when a target gene fragment exists in a reaction system, the primer probe is combined with the target gene, the fluorescent probe corresponding to the amplification stage is hydrolyzed, and no corresponding double strand is generated during the analysis of a melting curve, so that a specific peak cannot appear in the melting curve;
in conclusion: when a signal exists in the amplification stage and a corresponding peak disappears in the later stage melting curve analysis, the sample can be interpreted as a positive sample, and the disappeared peak represents a corresponding positive type.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
(1) The invention improves the influence of the number of the fluorescein on the channel of the fluorescence PCR method and improves the detection flux.
(2) The traditional microscopic examination of the plasmodium is a gold standard detection method, the microscopic examination has extremely high requirements on operators, the method is simple, quick and easy to train, and the requirements are avoided.
(3) The kit has high detection specificity on plasmodium, and solves the problem that the plasmodium poc/pow and pm/pk are difficult to distinguish in microscopic examination.
(4) The invention increases the sexual stage and asexual stage detection differentiation of the plasmodium falciparum, and can be used for epidemiological research on the plasmodium falciparum.
(5) The invention detects that other pathogenic microorganisms (dengue virus, neisseria meningitidis, haemophilus influenzae, toxoplasma, schistosome, streptococcus agalactiae and hantavirus) similar to malaria symptoms or at the same infection part and total nucleic acid of human are not cross-reacted
(6) The kit has high detection sensitivity, and can detect the plasmodium gene concentration of 1000copies/ml.
Drawings
FIG. 1 is a schematic diagram of a melting curve phase;
FIG. 2 is a schematic diagram of the detection of the present invention;
FIG. 3 is a result diagram of amplification curve and melting curve of the positive standard substance, wherein FIG. 3A is a result diagram of FAM channel amplification curve and melting curve, and FIG. 3B is a result diagram of VIC channel amplification curve and melting curve;
FIG. 4 is a result diagram of the ROX and CY5 channel amplification curves and melting curves of the positive standard, wherein FIG. 4A is a result diagram of the ROX channel amplification curve and melting curve, and FIG. 4B is a result diagram of the CY5 channel amplification curve and melting curve;
FIG. 5 shows the results of detecting sample 1 of example, in which a pf-a amplification curve and a melting curve were detected;
FIG. 6 shows the results of detecting sample 2 of example, in which a pf-s amplification curve and a melting curve were detected;
FIG. 7 shows the results of detecting sample 3 of example, in which a pv amplification curve and a melting curve were detected;
FIG. 8 shows the results of the detection of sample 4 of example, in which a pm amplification curve and a melting curve were detected;
FIG. 9 shows the results of detecting sample 5 of example, which detected a poc amplification curve and a melting curve;
FIG. 10 shows the results of detecting sample 6 of example, in which a pow amplification curve and a melting curve were detected;
FIG. 11 shows the results of detecting sample 7 of example, in which a pk amplification curve and a melting curve were detected;
FIG. 12 is a ROX channel amplification curve and a melting curve of samples 1 to 7 of the detection example;
FIG. 13 is a CY5 channel amplification curve and a melting curve of samples 1 to 7 of the detection example;
FIG. 14 is FAM channel amplification curves and melting curves of samples 8 to 14 of the detection examples;
FIG. 15 shows VIC channel amplification curves and melting curves of samples 8 to 14 of the detection examples;
FIG. 16 is ROX channel amplification curves and melting curves for samples 8-14 of the detection examples;
FIG. 17 is a CY5 channel amplification curve and a melting curve of samples 8 to 14 of the detection examples;
Detailed Description
The present invention is further explained with reference to the following examples, which are not intended to limit the present invention in any way.
Example 1 primer probe design:
downloading 18S ribosomal RNA genes of pf-a, pf-S, pv, poc, pow, pm and pk and human-derived Rnasep genes by NCBI search, designing primers and probes by using Primer Express 3.0.1 according to the technical principle, designing a plurality of groups of initial fp primers, then carrying out permutation and combination according to the volume ratio of fp: tp = 1.
Example 2 preparation of primer-probe mixture:
the preparation of the plasmodium melting curve detection solution is completed in the preparation of reagents in a PCR laboratory.
After multiple optimization, the formula of the plasmodium melting curve detection solution (primer probe and enzyme system) is shown in table 1:
TABLE 1
In the table, the volume of a single nucleic acid amplification reaction solution is 12.5. Mu.L, and the preparation method of the nucleic acid amplification reaction solution is as follows:
potassium chloride: 180mM, magnesium chloride: 45mM, ammonium sulfate: 80mM, dATP:20mM, dTTP:20mM, dGTP:20mM, dCTP:20mM, EDTA:2mM, taq DNA polymerase: 40U, regulating the pH value to 8.0 by using Tris-HCl, and taking glycerol as a solvent;
in this example, 14 samples from the respiratory disease control institute of Jiangsu province are to be examined, so that the preparation amount of the melting curve detection solution n =14+4+1=19 persons, where "14" represents the total amount of the samples detected at this time, "4" represents 4 positive controls in the positive standard, "1" represents 1 blank control, and the preparation of the melting curve detection solution in this example is performed as shown in Table 2:
TABLE 2
| Primer Probe name | Concentration of primer probes before |
1 part by |
19 parts by volume | Final concentration of reaction |
| fp1 | 100μM | 0.12μL | 2.28μL | 400nM |
| rp1 | 100μM | 0.12μL | 2.28μL | 400nM |
| tp1 | 100μM | 0.09μL | 1.71μL | 300nM |
| rp2 | 100μM | 0.12μL | 2.28μL | 400nM |
| tp2 | 10μM | 0.12μL | 2.28μL | 40nM |
| tp3 | 10μM | 0.09μL | 1.71μL | 30nM |
| tp4 | 10μM | 0.075μL | 1.425μL | 25nM |
| tp5 | 10μM | 0.09μL | 1.71μL | 30nM |
| tp6 | 10μM | 0.075μL | 1.425μL | 25nM |
| tp7 | 10μM | 0.075μL | 1.425μL | 25nM |
| tp8 | 10μM | 0.12μL | 2.28μL | 40nM |
| fp2 | 100μM | 0.12μL | 2.28μL | 400nM |
| fp3 | 100μM | 0.12μL | 2.28μL | 400nM |
| fp4 | 100μM | 0.12μL | 2.28μL | 400nM |
| fp5 | 100μM | 0.12μL | 2.28μL | 400nM |
| fp6 | 100μM | 0.12μL | 2.28μL | 400nM |
| fp7 | 100μM | 0.12μL | 2.28μL | 400nM |
| fp8 | 100μM | 0.12μL | 2.28μL | 400nM |
| fp9 | 100μM | 0.12μL | 2.28μL | 400nM |
| rp3 | 100μM | 0.12μL | 2.28μL | 400nM |
| tp9 | 100μM | 0.09μL | 1.71μL | 300nM |
| Nucleic acid amplification reaction solution | / | 12.5μL | 237.5μL | / |
| TE buffer solution | / | 5.235μL | 99.465μL | / |
| Total volume | / | 20μL | 380μL | / |
The plasmodium melting curve detection solution is respectively subpackaged into 19 hole sites of eight-row pipes according to the volume of 20 mu L per person, and the subpackaged eight-row pipes are transmitted to a sample processing room of a PCR laboratory.
Example 3 nucleic acid extraction:
extracting DNA in a sample to be detected by adopting a commercial whole blood extraction kit, and extracting a sample from a tin-free schistosomiasis prevention and treatment institute according to an instruction, wherein the template DNA extraction is required to be completed in a sample treatment room of a PCR laboratory; in this example, 14 template DNAs of samples to be tested were extracted, wherein samples 1 to 7 were derived from known Plasmodium positive patients, and samples 8 to 14 were derived from known Plasmodium negative patients.
Example 4 Standard preparation
The positive standard substances are in 4 groups, and are respectively:
positive control 1: the concentration ratio of the pf-a gene fragment to the poc gene fragment =10 6 copies/ml：10 6 Preparing copies/ml, and using TE buffer solution as solvent;
positive control 2: pf-s Gene fragment-pow Gene fragment = 2X 10 in concentration ratio 5 copies/ml：5×10 5 Preparing copies/ml, and using TE buffer solution as a solvent;
positive control 3: pm Gene fragment =10 as concentration ratio of pv Gene fragment to pm Gene fragment 6 copies/ml：8×10 5 Preparing copies/ml, and using TE buffer solution as solvent;
positive control 4: according to the concentration ratio, the pk gene fragment to the Rnasep gene fragment =10 6 copies/ml：10 7 copies/ml, and the solvent is TE buffer.
The blank standard is sterilized DNA-removed water.
EXAMPLE 5 nucleic acid Loading
The sample DNA extracted in example 3, the positive standard prepared in example 4, and the blank control were added to the eight-row calandria filled with the plasmodium melt curve detection solution described in example 1 in a volume of 10. Mu.L, respectively, so that the final reaction concentration of the primer probe set is as shown in Table 2, and the mixture was shaken and centrifuged.
Example 6 run on the machine and analysis:
the invention sets program on the real-time fluorescence PCR instrument according to the following content, and runs, the invention through testing and researching, compatible PCR instrument includes: ABI7500, quantstudio TM 5、Bio-Rad CFX96 TM The Shanghai macrostone SLAN-96P/S and Roche LightCycler480 II fluorescent quantitative PCR instruments were used in this example, the Shanghai macrostone SLAN-96S fluorescent quantitative PCR instrument was used.
Step (1): 95 ℃ for 10min (1 cycles);
step (2): 95 ℃ X10 s,58 ℃ X30s, 60 ℃ X10 s (40 cycles);
and (3): 95 ℃ X10 s,20-65 ℃ (0.03 ℃/s), 37 ℃ X10 s (1 cycles);
step (2) and step (3) at 58 ℃ for 30s and 20-65 ℃ (0.03 ℃/s), FAM, VIC, ROX and CY5 are arranged to collect fluorescence. The real-time fluorescent PCR instrument of ABI7500 and other instruments selects no ROX correction, and the quenching group selects None.
And (4) analyzing results:
firstly, analyzing a real-time fluorescent PCR amplification result, and directly judging that a sample to be detected is negative if FAM, VIC and ROX channel amplification results are negative and CY5 channel amplification is positive;
if the ROX channel amplification result is positive, the sample to be detected is plasmodium positive, at least one of the FAM and VIC channels is also positive, then melting curve analysis is carried out in the fluorescence channel with the FAM and VIC corresponding amplification results positive, the specific type of the plasmodium is judged, and the type of the plasmodium corresponding to the disappeared peak is the positive type. The specific analysis can refer to the operation instruction of the corresponding fluorescent PCR instrument.
Example 7 interpretation of results:
positive judgment value: the method comprises the steps of detecting critical positive samples and critical negative samples, drawing an ROC curve by adopting a statistical method, and determining a positive judgment value by adopting a Youden index (Youden index);
wherein, the Ct of the detection result in the embodiment 7 is less than or equal to 38, and the result is judged to be positive when the melting curve map has obvious difference with a blank reference substance;
the detection result Ct is more than 38 or the negative result is judged when the detection result is not detected;
the target genes of FAM channel detection are pf-a, pf-s and pv, and three specific main peaks which sequentially appear in a temperature increasing manner respectively represent pf-a, pf-s and pv during melting curve analysis;
the target genes of VIC channel detection are pm, poc, pow and pk, and four specific main peaks which sequentially appear in a temperature increasing manner respectively represent pm, poc, pow and pk during melting curve analysis;
the target gene of the ROX channel detection is plasmodium universal gene which can infect human,
the target gene of CY5 channel detection is Rnasep;
the detection of FAM and VIC channels needs to be combined with melting curve analysis and interpretation, ROX and CY5 channels do not carry out melting curve analysis, the ROX channel only interprets the positive and negative plasmodium of a sample to be detected through a real-time fluorescence PCR amplification curve result, and the CY5 channel only interprets the positive and negative internal standards of the sample to be detected through the real-time fluorescence PCR amplification curve result;
the results show that:
the positive standard FAM channel melting curve results show (fig. 3A from left to right):
FIG. 3A-1: the dark line is the pf-a detection result, the light line represents a negative control, the lower graph is an amplification result, the Ct is 21.94, the Ct is less than or equal to 38, and the melting curve map has significant difference with a blank control, which indicates that pf-a is positive in the amplification of the FAM channel, the upper graph is the melting curve result, wherein the first peak (Tm =33 +/-1 ℃) disappears from left to right, and the two comprehensively represent that pf-a is positive;
FIGS. 3A-2: the dark line is the pf-s detection result, the light line represents a negative control, the lower graph is an amplification result, the Ct of the amplification result is 25.63, the Ct is less than or equal to 38, and the melting curve map has significant difference with a blank control, which indicates that pf-s is positive in amplification of an FAM channel, the upper graph is a melting curve result, wherein the second peak (Tm =44 +/-1 ℃) disappears from left to right, and the two comprehensively represent that pf-s is positive;
FIGS. 3A-3: the dark line is the result of pv detection, the light line represents negative control, the lower graph is the amplification result, the Ct is 23.47, the Ct is less than or equal to 38, and the melting curve map is significantly different from the blank control, which indicates that pv is positive in amplification in the FAM channel, the upper graph is the melting curve result, wherein the third peak from left to right (Tm =56 ± 1 ℃) disappears, and the two are combined to represent pv positive.
The result of the VIC channel melting curve of the positive standard is shown (fig. 3B from left to right):
FIG. 3B-1: the dark line is the pm detection result, the light line represents the negative control, the lower graph is the amplification result, the Ct is 15.12, the Ct is less than or equal to 38, the melting curve spectrum is obviously different from the blank control, the pm is amplified positively in the VIC channel, the upper graph is the melting curve result, the first peak (Tm =29 +/-1 ℃) disappears from left to right, and the two are combined to represent the pm is positive.
FIG. 3B-2: the dark line is the poc detection result, the light line represents the negative control, the lower graph is the amplification result, the Ct is 13.06, the Ct is less than or equal to 38, and the melting curve spectrum is significantly different from the blank control, which indicates that the poc is amplified in the VIC channel to be positive, the upper graph is the melting curve result, wherein the second peak (Tm =41 +/-1 ℃) disappears from left to right, and the two are combined to represent the poc to be positive.
FIGS. 3B-3: the dark line is the result of the pow detection, the light line represents the negative control, the lower graph is the amplification result, the Ct is 17.57, the Ct is less than or equal to 38, and the melting curve spectrum is significantly different from the blank control, which indicates that the amplification of the pow in the VIC channel is positive, the upper graph is the result of the melting curve, wherein the third peak from left to right (Tm =49 +/-1 ℃) disappears, and the two are combined to represent the positive pow.
FIGS. 3B-4: the dark line is the pk detection result, the light line represents the negative control, the lower graph is the amplification result, the Ct is 20.93, the Ct is less than or equal to 38, and the melting curve map is significantly different from the blank control, which indicates that pk is positive in the VIC channel amplification, the upper graph is the melting curve result, wherein the fourth peak from left to right (Tm =58 ± 1 ℃) disappears, and the two are combined to represent pk positive.
Positive standard ROX channel melting curve results show (fig. 4A): the ROX channel does not perform melting curve analysis, only an amplification curve below the image is analyzed, and the amplification curve analysis shows that the plasmodium positive standard substances to be detected (positive control 1, positive control 2, positive control 3 and positive control 4) all have amplification curves in the ROX channel, so that the result can be judged to be positive when the positive standard substances are detected.
Positive standard CY5 channel melting curve results show (fig. 4B): and (3) analyzing only an amplification curve below the map without performing melting curve analysis on the CY5 channel, and analyzing the amplification curve, wherein only the positive control 4 has an amplification curve in the CY5 channel in the plasmodium positive standard products to be detected (the positive control 1, the positive control 2, the positive control 3 and the positive control 4), so that the normal amplification of the internal standard of the positive control 4 can be judged.
The results of the samples to be detected are as follows:
FIG. 5 is a lower graph of the FAM channel real-time fluorescence PCR detection result of the sample 1 to be detected, the Ct of the FAM channel real-time fluorescence PCR detection result is 16.23, the Ct is less than or equal to 38, and the melting curve map has significant difference (within a dotted circle) from a blank reference, and the FAM channel real-time fluorescence PCR detection result shows that the sample to be detected is positive for plasmodium; FIG. 5 is a FAM channel melting curve of the sample 1, the first peak from left to right being the pf-a peak, and the disappearance of the peak in FIG. 5 indicates the detection of pf-a by the organism to be detected;
FIG. 6 is a lower graph of the FAM channel real-time fluorescence PCR detection result of the sample 2 to be detected, the Ct of the FAM channel real-time fluorescence PCR detection result is 19.35, the Ct is less than or equal to 38, and the melting curve map has significant difference (within a dotted circle) from a blank reference, and the FAM channel real-time fluorescence PCR detection result shows that the sample to be detected is positive for plasmodium; the upper graph in FIG. 6 is the FAM channel melting curve of the sample 2 to be detected, the second peak from left to right is the pf-s peak, and the disappearance of the peak in FIG. 6 indicates the detection of pf-s by the organism to be detected;
FIG. 7 is a lower graph of the FAM channel real-time fluorescence PCR detection result of the sample 3 to be detected, the Ct of the FAM channel real-time fluorescence PCR detection result is 27.84, the Ct is less than or equal to 38, and the melting curve map has significant difference (within a dotted circle) from a blank reference, and the FAM channel real-time fluorescence PCR detection result shows that the sample to be detected is positive for plasmodium; the upper graph in FIG. 7 is a FAM channel melting curve of the sample to be detected 3, the third peak from left to right is the pv peak, and the disappearance of the peak in FIG. 7 indicates that the pv is detected by the organism to be detected;
FIG. 8 shows the lower graph of the real-time fluorescence PCR detection result of the VIC channel of the sample 4 to be detected, the Ct of which is 21.89, the Ct is less than or equal to 38, and the melting curve spectrum has significant difference (within the dotted circle) from the blank control, showing that the sample is positive to plasmodium; the upper graph in FIG. 8 is a VIC channel melting curve of the sample 4 to be detected, the first peak from left to right is the pm peak, and the disappearance of the pm peak in FIG. 8 indicates that the pm is detected by the organism to be detected;
FIG. 9 is a lower graph of the real-time fluorescence PCR detection result of the VIC channel of the sample 5 to be detected, wherein the Ct is 21.17, the Ct is less than or equal to 38, and the melting curve spectrum has significant difference (within a dotted circle) from a blank reference, and shows that the sample to be detected is positive to plasmodium; FIG. 9 is a top view of a VIC channel melting curve of a sample 5 to be tested, showing a second peak from left to right, namely a poc peak, and showing that the poc peak is detected by a biological sample to be tested when the peak disappears in FIG. 9;
FIG. 10 is a lower graph of the real-time fluorescence PCR detection result of the VIC channel of the sample 6 to be detected, the Ct of the real-time fluorescence PCR detection result is 17.58, the Ct is less than or equal to 38, and the melting curve spectrum has significant difference (within a dotted circle) from a blank control, and the real-time fluorescence PCR detection result shows that the sample to be detected is positive for plasmodium; the upper graph in FIG. 10 is a VIC channel melting curve of the sample to be tested 6, and the third peak from left to right, namely the pow peak, and the disappearance of the peak in FIG. 10 indicates the detection of the pow by the organism to be tested;
FIG. 11 is a lower graph of the real-time fluorescence PCR detection result of the VIC channel of the sample 7 to be detected, the Ct of the real-time fluorescence PCR detection result is 26.86, the Ct is less than or equal to 38, and the melting curve spectrum has significant difference (within a dotted circle) from a blank control, and the real-time fluorescence PCR detection result shows that the sample to be detected is positive for plasmodium; FIG. 11 is a top view of a VIC channel melting curve of the sample 7, showing that the fourth peak from left to right is the pk peak, and the disappearance of this peak in FIG. 11 indicates the detection of pk by the organism to be detected;
FIG. 12 is a lower graph of ROX channel real-time fluorescence PCR detection results of a sample to be detected 1, a sample to be detected 2, a sample to be detected 3, a sample to be detected 4, a sample to be detected 5, a sample to be detected 6 and a sample to be detected 7, wherein Ct of the real-time fluorescence PCR detection results is 16.61, 20.04, 31.14, 20.16, 20.18, 12.55 and 24.73 in sequence, ct is less than or equal to 38, and the result shows that the sample to be detected is positive to plasmodium; FIG. 12 is a top view showing ROX channel melting curves of a sample to be examined 1, a sample to be examined 2, a sample to be examined 3, a sample to be examined 4, a sample to be examined 5, a sample to be examined 6, and a sample to be examined 7, without any analysis, and thus FIG. 12 shows that the sample to be examined 1, the sample to be examined 2, the sample to be examined 3, the sample to be examined 4, the sample to be examined 5, the sample to be examined 6, and the sample to be examined 7 are positive for Plasmodium; the detection result corresponds to the actual situation.
FIG. 13 shows the CY5 channel real-time fluorescence PCR detection results of a sample to be detected 1, a sample to be detected 2, a sample to be detected 3, a sample to be detected 4, a sample to be detected 5, a sample to be detected 6 and a sample to be detected 7, wherein Ct of the real-time fluorescence PCR detection results is 15.79, 14.76, 19.84, 17.03, 17.62, 15.71 and 15.32 in sequence, ct is less than or equal to 38, and the result shows that the internal standard of the sample to be detected is positive; the upper diagram of fig. 13 is the CY5 channel melting curve of the sample to be detected 1, the sample to be detected 2, the sample to be detected 3, the sample to be detected 4, the sample to be detected 5, the sample to be detected 6 and the sample to be detected 7, and the channel melting curve is not analyzed, so that fig. 13 shows that the internal standards of the sample to be detected 1, the sample to be detected 2, the sample to be detected 3, the sample to be detected 4, the sample to be detected 5, the sample to be detected 6 and the sample to be detected 7 are qualified in detection, and the detection result is credible.
FIG. 14 shows FAM channel real-time fluorescence PCR detection results and melting curves of a sample to be detected 8, a sample to be detected 9, a sample to be detected 10, a sample to be detected 11, a sample to be detected 12, a sample to be detected 13, and a sample to be detected 14; the amplification curves of the lower graph of FIG. 14 are all negative, and the melting curves of the upper graph of FIG. 14 are complete, and comprehensively show that the sample to be detected 8, the sample to be detected 9, the sample to be detected 10, the sample to be detected 11, the sample to be detected 12, the sample to be detected 13 and the sample to be detected 14 are all negative in the FAM channel.
FIG. 15 shows the VIC channel real-time fluorescence PCR detection results and melting curves of a sample 8 to be detected, a sample 9 to be detected, a sample 10 to be detected, a sample 11 to be detected, a sample 12 to be detected, a sample 13 to be detected, and a sample 14 to be detected; the amplification curves of the lower graph of FIG. 15 are all negative, and the melting curves of the upper graph of FIG. 15 are complete, which comprehensively shows that the sample 8 to be tested, the sample 9 to be tested, the sample 10 to be tested, the sample 11 to be tested, the sample 12 to be tested, the sample 13 to be tested and the sample 14 to be tested are all negative in the VIC channel.
FIG. 16 shows the ROX channel real-time fluorescence PCR detection results of sample 8 to be detected, sample 9 to be detected, sample 10 to be detected, sample 11 to be detected, sample 12 to be detected, sample 13 to be detected, and sample 14 to be detected, and the amplification curves are negative; the upper diagram of FIG. 14 shows, without melting curve analysis, that the sample to be examined 8, the sample to be examined 9, the sample to be examined 10, the sample to be examined 11, the sample to be examined 12, the sample to be examined 13, and the sample to be examined 14 are all negative in the ROX channel.
FIG. 17 shows the CY5 channel real-time fluorescence PCR detection results of a sample to be detected 8, a sample to be detected 9, a sample to be detected 10, a sample to be detected 11, a sample to be detected 12, a sample to be detected 13 and a sample to be detected 14, the Ct values of which are 14.39, 15.82, 18.64, 11.48, 11.83, 19.16 and 16.44 in sequence, the Ct value is less than or equal to 38, and the result shows that the internal standard of the sample to be detected is positive; the upper diagram of fig. 14 does not perform melting curve analysis, and comprehensively shows that the internal standard detection of the sample 8 to be detected, the sample 9 to be detected, the sample 10 to be detected, the sample 11 to be detected, the sample 12 to be detected, the sample 13 to be detected, and the sample 14 to be detected is normal, and the results of fig. 14, fig. 15, and fig. 16 can be combined to judge that the sample 8, the sample 9 to be detected, the sample 10 to be detected, the sample 11 to be detected, the sample 12 to be detected, the sample 13 to be detected, and the sample 14 to be detected are true negative results, but not false negative results caused by sampling, extraction or operation.
The 14 samples are 14 samples from the institute for prevention and treatment of schistosomiasis casseroides, jiangsu, and whether the samples are positive or not, and the information of the specific positive type and the like is consistent with the detection result. The detection accuracy is 100%.
Example 8 Performance Studies
And (3) specific analysis:
the detection methods described in examples 2 to 8 were used to detect other pathogenic microorganisms (dengue virus, neisseria meningitidis, haemophilus influenzae, toxoplasma, schistosome, streptococcus agalactiae, hantavirus) from the stanniferous schistosomiasis prevention and treatment institute, which are similar to malaria symptoms or have the same infection site, and human total nucleic acids, and the detection results were all negative, indicating that the method has excellent specificity.
And (3) confirming the lowest detection limit:
quantifying a plasmodium sample from a tin-free schistosomiasis prevention and treatment research institute by using a digital PCR instrument, then respectively diluting plasmodium DNA with known concentration to 5000copies/ml, 1000copies/ml and 800copies/ml by using TE buffer solution for detection, wherein the detection result shows that when the concentration is 5000copies/ml, the detection rate of all types is 100%; when the concentration is 1000copies/ml, only the plasmodium vivax meets the detection rate of 95% (19/20), and the detection rate of other types is 100% (20/20), so the minimum detection limit of the invention is defined as 1000copies/ml.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (11)
1. A primer probe combination for detecting and parting 7 plasmodia, which is characterized in that the 7 plasmodia are plasmodium falciparum pf-a in asexual stage, plasmodium falciparum pf-s in sexual stage, plasmodium vivax pv, plasmodium ovale poc subtype, plasmodium ovale pow subtype, plasmodium malariae pm, plasmodium knowlesi pk;
the primer probe combination comprises:
universal primers and probes fp1, rp1 and tp1 for detecting plasmodium in human body;
a primer rp2 for distinguishing the plasmodium types of the human bodies;
probes tp2, tp3, tp4, tp5, tp6, tp7 and tp8 for distinguishing plasmodium types of human bodies;
melting curve primers fp2, fp3, fp4, fp5, fp6, fp7 and fp8 for distinguishing plasmodium types of human bodies;
internal reference gene primers and probes fp9, rp3 and tp9 for monitoring the reaction process;
the fp1 nucleotide sequence is shown as SEQ ID NO. 1;
the rp1 nucleotide sequence is shown in SEQ ID NO. 2';
the tp1 nucleotide sequence is shown as SEQ ID NO.3, ROX fluorescein is marked at the 5 'part of the tp1, and MGB fluorescence quenching groups are marked at the 3' part of the tp1;
the rp2 nucleotide sequence is shown as SEQ ID NO. 4;
the tp2 nucleotide sequence is shown as SEQ ID NO.5, FAM fluorescein is marked at the 5 'of the tp2, and MGB fluorescence quenching groups are marked at the 3' of the tp 2;
the tp3 nucleotide sequence is shown as SEQ ID NO.6, FAM fluorescein is marked at the 5 'part of the tp3, and MGB fluorescence quenching groups are marked at the 3' part of the tp 3;
the tp4 nucleotide sequence is shown as SEQ ID NO.7, FAM fluorescein is marked at the 5 'part of the tp4, and MGB fluorescence quenching groups are marked at the 3' part of the tp 4;
the tp5 nucleotide sequence is shown as SEQ ID NO.8, VIC fluorescein is marked at the 5 'part of the tp5, and MGB fluorescence quenching groups are marked at the 3' part of the tp 5;
the tp6 nucleotide sequence is shown as SEQ ID NO.9, VIC fluorescein is marked at the 5 'part of the tp6, and MGB fluorescence quenching groups are marked at the 3' part of the tp 6;
the tp7 nucleotide sequence is shown as SEQ ID NO.10, VIC fluorescein is marked at the 5 'part of the tp7, and MGB fluorescence quenching groups are marked at the 3' part of the tp 7;
the tp8 nucleotide sequence is shown as SEQ ID NO.11, VIC fluorescein is marked at the 5 'part of the tp8, and MGB fluorescence quenching groups are marked at the 3' part of the tp8 nucleotide sequence;
the fp2 nucleotide sequence is shown as SEQ ID NO. 12;
the fp3 nucleotide sequence is shown as SEQ ID NO. 13;
the fp4 nucleotide sequence is shown as SEQ ID NO. 14;
the fp5 nucleotide sequence is shown as SEQ ID NO. 15;
the fp6 nucleotide sequence is shown as SEQ ID NO. 16;
the fp7 nucleotide sequence is shown as SEQ ID NO. 17;
the fp8 nucleotide sequence is shown as SEQ ID NO. 18;
the fp9 nucleotide sequence is shown as SEQ ID NO.19
The rp3 nucleotide sequence is shown as SEQ ID NO. 20;
the tp9 nucleotide sequence is shown as SEQ ID NO.21, CY5 fluorescein is marked at the 5 'end of the tp9, and a BHQ3 fluorescence quenching group is marked at the 3' end of the tp9.
2. A kit for detecting and typing 7 plasmodia, comprising the primer probe combination of claim 1.
3. The kit for detecting and typing 7 plasmodium species according to claim 2, wherein the final concentration of the primer probe reaction in the kit is fp1:400nm, rp1 400nm, tp1, rp2, 400nm, tp2.
4. The kit for detecting and typing 7 plasmodia according to claim 2, wherein the kit further comprises a nucleic acid amplification reaction solution, a positive standard, a blank control and a TE buffer solution.
5. The kit for detecting and typing 7 plasmodia according to claim 4, wherein the nucleic acid amplification reaction solution comprises a mixture of one or more of tris, potassium chloride, magnesium chloride, ammonium sulfate, dNTP, and Taq DNA polymerase.
6. The kit for detecting and typing 7 plasmodium according to claim 5, wherein the nucleic acid amplification reaction solution comprises potassium chloride: 180mM, magnesium chloride: 45mM, ammonium sulfate: 80mM, dATP:20mM, dTTP:20mM, dGTP:20mM, dCTP:20mM, EDTA:2mM, taq DNA polymerase: 40U, pH adjusted to 8.0 using Tris-HCl and glycerol as solvent.
7. The kit for detecting and typing 7 plasmodia according to claim 4, wherein the positive standard comprises: a sexual stage plasmodium falciparum pf-s gene segment with a nucleotide sequence shown as SEQ ID No.22, a asexual stage plasmodium falciparum pf-a gene segment with a nucleotide sequence shown as SEQ ID No.23, a plasmodium vivax pv gene segment with a nucleotide sequence shown as SEQ ID No.24, a plasmodium ovale poc gene segment with a nucleotide sequence shown as SEQ ID No.25, a plasmodium ovale pow gene segment with a nucleotide sequence shown as SEQ ID No.26, a plasmodium malariae pm gene segment with a nucleotide sequence shown as SEQ ID No.27, a plasmodium knowlesi pk gene segment with a nucleotide sequence shown as SEQ ID No.28 and a ribonuclease Rnasep gene segment with a nucleotide sequence shown as SEQ ID No. 29.
8. The kit for detecting and typing 7 plasmodia according to claim 7, wherein the positive standard comprises positive control 1, positive control 2, positive control 3, positive control 4; the preparation method specifically comprises the following steps:
positive control 1: the concentration ratio of the pf-a gene fragment to the poc gene fragment =10 6 copies/ml：10 6 Preparing copies/ml, and using TE buffer solution as solvent;
positive control 2: pf-s Gene fragment-pow Gene fragment = 2X 10 in concentration ratio 5 copies/ml：5×10 5 Preparing copies/ml, and using TE buffer solution as a solvent;
positive control 3: pm gene fragment according to the concentration ratioGene fragment =10 6 copies/ml：8×10 5 Preparing copies/ml, and using TE buffer solution as a solvent;
positive control 4: according to the concentration ratio, the pk gene fragment to the Rnasep gene fragment =10 6 copies/ml：10 7 copies/ml, and the solvent is TE buffer.
9. The kit for detecting and typing 7 plasmodia according to claim 4, wherein the blank control is sterilized and DNA-removed water.
10. Use of the primer probe combination of claim 1 for preparing reagents for detecting and typing 7 plasmodium species.
11. A method for detecting and typing 7 plasmodia, comprising the steps of:
step 1, preparing a plasmodium melting curve detection solution:
step 1-1: calculating a preparation amount n, wherein n = detection person +4 person positive standard substance +1 person blank reference substance;
step 1-2: preparing a plasmodium melting curve detection solution: adding the primer probe combination of claim 1 into a nucleic acid amplification reaction solution, adjusting the volume by using a TE buffer solution, and preparing a plasmodium melting curve detection solution, wherein the preparation part of the plasmodium melting curve detection solution is not less than n;
step 2, extracting template DNA:
extracting DNA from blood of an organism to be detected as template DNA;
step 3, nucleic acid sample adding:
the template DNA extracted in step 2, the positive standard, and the blank control are added to the split plasmodium melt curve test solution described in step 1, respectively, so that the final reaction concentration of the primer-probe combination described in claim 1 is fp1:400nm, rp1 400nm, tp1; oscillating and centrifuging;
and 4, program operation:
and (3) operating the system obtained in the step (3) on a real-time fluorescent PCR instrument, wherein the operating program is as follows:
step (1): multiplying by 10min at 95 ℃;1cycles
Step (2): 95 ℃ X10 s,58 ℃ X30s, 60 ℃ X10 s;40cycles
And (3): 95 ℃ multiplied by 10s,20-65 ℃ multiplied by 10s, 37 ℃;1cycles
Step (2) and step (3) of collecting fluorescence by setting FAM, VIC, ROX and CY5 at 58 ℃ for 30s and 20-65 ℃; real-time fluorescent PCR is not selected for ROX correction, and a quenching group is selected for None;
step (3) and the temperature of 20-65 ℃ is the temperature rise process of the melting curve, and the temperature rise rate is 0.03 ℃/s.
And 5, analyzing results:
firstly, analyzing a real-time fluorescent PCR amplification result, and directly judging that a sample to be detected is negative if FAM, VIC and ROX channel amplification results are negative and CY5 channel amplification is positive;
if the ROX channel amplification result is positive, the sample to be detected is plasmodium positive, at least one of the FAM and VIC channels is also positive, then melting curve analysis is carried out in the fluorescence channel with the FAM and VIC corresponding amplification results positive, the specific type of the plasmodium is judged, and the type of the plasmodium corresponding to the disappeared peak is the positive type;
6, judging the real-time fluorescent PCR amplification result in the step 5:
positive judgment value:
when the Ct of the detection result is less than or equal to 38 and the melting curve map is significantly different from the blank control, the amplification result is judged to be positive; when the detection result Ct is more than 38 or is not detected, the amplification result is judged to be negative;
and (3) positive type judgment:
the target genes of FAM channel detection are pf-a, pf-s and pv, and three specific main peaks which sequentially appear in a temperature increasing manner respectively represent pf-a, pf-s and pv during melting curve analysis;
the target genes of VIC channel detection are pm, poc, pow and pk, and four specific main peaks which sequentially appear in a temperature increasing manner respectively represent pm, poc, pow and pk during melting curve analysis;
the target gene of the ROX channel detection is plasmodium universal gene which can infect human,
the target gene of CY5 channel detection is Rnasep;
the FAM and VIC channel detection needs to be combined with melting curve analysis and interpretation, the ROX and CY5 channels do not carry out melting curve analysis, and the negative and positive plasmodium of a sample to be detected is interpreted only through a real-time fluorescence PCR amplification result.
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