CN114277127A - Primer group, probe and kit for detecting Kawasaki disease - Google Patents
Primer group, probe and kit for detecting Kawasaki disease Download PDFInfo
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Abstract
The invention provides a primer group, a probe and a kit for detecting Kawasaki disease, belonging to the technical field of molecular biology, and comprising a reverse transcription primer, an amplification primer and a probe sequence, wherein the nucleic acid sequence combination can effectively qualitatively/quantitatively detect miR-125a-5p, hsa-miR-3182, hsa-miR-3675-5p, miR-4433b-5p and miR-126 in human blood platelets, and the miR-126 is used as an internal reference marker. The miRNA in the platelets is collected, and the platelets are easier to obtain and rich in content compared with exosomes, and the extraction method is simple. The invention sets specific miRNA reference genes, the method is more reasonable, 4 miRNAs are analyzed in a combined manner, the accuracy is far higher than that of detection of a single biological standard substance, the occurrence of false positive is reduced, and the situations of Kawasaki disease, common fever and the like are more easily distinguished.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to the technical field of miRNA application, and particularly relates to a primer group, a probe and a kit for detecting Kawasaki disease.
Background
Kawasaki Disease (KD) was first reported in 1967 by Kawasaki richness in japan and was called mucocutaneous lymph node syndrome (MCLS), an acute, self-limiting systemic immune vasculitis of unknown cause. It is characterized by extensive middle and small vasculitis, with the most severe damage to the cardiovascular system, mainly involving the coronary arteries, and approximately 15-25% of untreated children eventually develop coronary artery lesions, making it the most common cause of acquired heart disease in children in developed countries. Research suggests that long-term vascular endothelial dysfunction exists in both the acute phase and the convalescent phase of KD, and the vascular endothelial dysfunction becomes a risk factor for susceptibility to coronary atherosclerosis of adults. The 2004 american heart disease association proposed: persistent dysfunction of vascular endothelial function after KD recovery may be a new risk factor for coronary heart disease.
Kawasaki disease, an acute, self-limiting systemic vasculitis, is characterized clinically by fever, changes in the oral mucosa, rash, enlargement of the cervical lymph nodes, redness of the bulbar conjunctiva and changes in the extremities. It is characterized by extensive medium and small angiopathy, and the damage to cardiovascular system is most serious, and the histopathological manifestations include whole vasculitis, endothelial necrosis, mononuclear cell infiltration of medium and small blood vessels, etc. If the treatment is not regularly performed in time, coronary artery lesion appears in 25 to 30 percent of children patients; and the incidence of coronary aneurysm is reduced to about 3-5% by applying intravenous large dose gamma globulin (IVIG) in the early stage. The early stage of the Kawasaki disease is mainly characterized in that the heat course of high patients exceeds 5 days, and the high patients are easily confused with other febrile diseases such as cervical lymphadenitis, scarlet fever, urticaria, septicemia and the like to generate misdiagnosis, and according to related literature reports, the early stage misdiagnosis rate of the Kawasaki disease can reach 26.7%, so that the misdiagnosed children cannot use IVIG for combined treatment in the early stage of the Kawasaki disease in time to cause later-stage coronary artery injury.
Platelets, the second most abundant cell type in blood, are produced by megakaryocytes in bone marrow hematopoietic tissues. The multifunctional hemopoietic stem cell is directionally differentiated in hemopoietic tissue to form primitive megakaryocyte, which is further become mature megakaryocyte. The surface of mature megakaryocyte membrane forms many pits to extend into cytoplasm, and adjacent pit membranes are fused with each other in the deep pits to separate the cytoplasm of megakaryocyte from mother body. Finally, the components separated from the megakaryocyte cytoplasm and surrounded by the cell membrane are separated from the megakaryocytes, pass through the blood sinuses in the hematopoietic tissue of the bone marrow and enter the blood circulation to become platelets. Platelets, which are present in the peripheral blood circulation, play a role in the body in hemostasis and wound healing.
In recent years, it has been found that platelets become central molecules of systemic and local reactions, play an important role in cell communication and immune reactions, and since platelets have no nuclei, there is no background interference of rRNA in the study of their RNA. The platelets also play roles in signal exchange and transfer propagation in immune response, and as Kawasaki disease is a systemic immune disease, the research on platelet RNA has theoretical basis and practical significance for early diagnosis of Kawasaki disease, but the current research reports on the aspect are very rare.
The patent CN 106701962A provides a primer group, a probe and a kit for detecting Kawasaki disease, and the primer group, the probe and the kit for detecting Kawasaki disease comprise reverse transcription primers, amplification primers and probe sequences, and the nucleic acid sequence combination can effectively detect hsa-miR-197, hsa-miR-671, hsa-miR1246 and hsa-miR4436 in human serum qualitatively/quantitatively. This patent acquires exosome miRNA from serum sample and detects, and serum in the blood acquires needs wait for a certain time, and exosome itself is difficult to acquire, and exosome miRNA's content is less, extracts exosome miRNA operation complicacy, and the extraction cost is higher, and exosome miRNA's the detection degree of difficulty is big. The reference gene is not set in the patent, and individual differences may exist in later experiments.
The patent CN104450901A discloses that hsa-miR-197, hsa-miR-671, hsa-miR-1246 and hsa-miR-4436 can be used as molecular markers of Kawasaki disease, and specifically discloses dye-process fluorescent quantitative PCR primer sequences of miR-1246, miR-4436b-5p, miR-197-3p and miR-671-5p, and 4 groups of 8 nucleic acid sequences in total, and the detection precision of the primers still needs to be improved.
Disclosure of Invention
The invention aims to provide a primer group, a probe and a kit for detecting Kawasaki disease, wherein miRNA in platelets is collected, and the platelets are easier to obtain compared with exosomes, rich in content and simple in extraction method. The invention sets specific miRNA reference genes, and the method is more reasonable. The 4 selected miRNAs are from the results of second-generation high-depth sequencing and obtained by combining clinical information comprehensive analysis scoring, and the 4 miRNAs are analyzed in a combined manner, so that the accuracy is far higher than that of detection of a single biological standard substance, and the occurrence of false positive is reduced. The Kawasaki disease and the common fever are more easily distinguished. Has the advantages of convenient material acquisition, low cost, high sensitivity, good stability, easy operation and the like.
The technical scheme of the invention is realized as follows:
the invention provides a nucleic acid sequence combination for detecting Kawasaki disease, which comprises a reverse transcription primer, an amplification primer and a probe sequence, and can effectively detect miR-125a-5p, hsa-miR-3182, hsa-miR-3675-5p, miR-4433b-5p and miR-126 in human blood platelets qualitatively/quantitatively, wherein miR-126 is used as an internal reference marker;
wherein, the reverse transcription primer sequence is as follows:
miR-125a-5p-sl：CAGGGCATCAGCCTGAACCCTGAACCCTGAATAACCTG AAACTGATGCCCTGTCACAG；
hsa-miR-3182-sl：ACGGGAAACTGAGTTCAGTTCCCAGCGTCCATATCAGT TCCCAGTTTCCCGTGACTAC；
hsa-miR-3675-5p-sl：CAGGGCATCAGCCTGAACCCTGAACCCTGAATAAC CTGAAACTGATGCCCTGGAAATC；
miR-4433b-5p-sl：CTTGATGGCTGCCCGTCAAGCCAGTCAAGTATCCAGT CAAGTCAGCCATCAAGACAGGA；
miR-126-sl：TTAGGGTCAGGCCTGAACCCTGAACCCTGAATGCGAACTG ACCTGACCCTAACGCATT；
the amplification primer sequences are as follows:
a forward primer:
miR-125a-5p-F：CCTGAACCCTGAACCCTGA；
hsa-miR-3182-F：AGTTCAGTTCCCAGCGTCC；
hsa-miR-3675-5p-F：CCTGAACCCTGAACCCTGA；
miR-4433b-5p-F：CCCGTCAAGCCAGTCAAGT；
miR-126-F：CCTGAACCCTGAACCCTGA；
reverse primer:
miR-125a-5p-R：GGTCCCTGAGACCCTTTAAC；
hsa-miR-3182-R：TGCGCGCTTCTGTAGT；
hsa-miR-3675-5p-R：CCTCTATGGGGCTTCTGTAGA；
miR-4433b-5p-R：GATGTCCCACCCCCAC；
miR-126-R：GAGCTCGTACCGTGAGTAAT；
the probe sequence is as follows:
miR-125a-5p-P：AACCTGAAACTGATGCCCTG；
hsa-miR-3182-P：ATCAGTTCCCAGTTTCCCGT；
hsa-miR-3675-5p-P：AACCTGAAACTGATGCCCTG；
miR-4433b-5p-P：CCAGTCAAGTCAGCCATCAAG；
miR-126-P：GCGAACTGACCTGACCCTAA。
the invention further provides a kit for detecting Kawasaki disease, which comprises a reverse transcription reagent, an amplification reagent and the probe sequence, wherein the 5 'end of the probe nucleotide sequence is marked with a fluorescence reporter group, the 3' end of the probe nucleotide sequence is marked with a fluorescence quenching group, the fluorescence reporter group of the probe nucleotide sequence is at least one of FAM, HEX, VIC, ROX and Cy5, and the fluorescence quenching group is at least one of BHQ1, BHQ2 and MGB; the reverse transcription primer sequence is the reverse transcription primer; the amplification primers are the forward primer and the reverse primer.
The invention further protects a detection chip or device, which comprises the reverse transcription primer, the amplification primer and the probe sequence.
The invention further protects the application of the reverse transcription primer, the amplification primer and the probe sequence in the preparation of reagents or tools for predicting and assisting in diagnosing the Kawasaki disease.
The invention further protects the application of the nucleic acid sequence combination in the preparation of reagents for qualitatively and/or quantitatively detecting miR-125a-5p, hsa-miR-3182, hsa-miR-3675-5p, miR-4433b-5p and miR-126.
The invention further provides a method for detecting target genes miR-125a-5p, hsa-miR-3182, hsa-miR-3675-5p, miR-4433b-5p and reference gene miR-126, which comprises the following steps:
1) extracting platelet miRNA from a sample, adding reverse transcription primers miR-125a-5p-sl, hsa-miR-3182-sl, hsa-miR-3675-5p-sl, miR-4433b-5p-sl and miR-126-sl for reverse transcription to obtain cDNA;
2) using cDNA as a template, performing amplification reaction by using a forward primer and a reverse primer, adding a probe sequence during the amplification reaction, performing RT-qPCR analysis on an amplification product, judging the Ct value of the fluorescence quantitative reaction of miRNA of a target gene and an internal reference gene, and calculating the expression quantity R of a nucleic acid marker;
the forward primer comprises miR-125a-5p-F, hsa-miR-3182-F, hsa-miR-3675-5p-F, miR-4433b-5p-F, miR-126-F;
the reverse primer comprises miR-125a-5p-R, hsa-miR-3182-R, hsa-miR-3675-5p-R, miR-4433b-5p-R, miR-126-R;
the probe sequence comprises miR-125a-5P-P, hsa-miR-3182-P, hsa-miR-3675-5P-P, miR-4433b-5P-P, miR-126-P;
the reverse transcription primers, amplification primers and probe sequences are as described above.
As a further improvement of the invention, the amplification reaction system and conditions are as follows:
Premix Ex Taq（Probe qPCR）（2X）：10μl；
primer: F/R is 0.4 mu l each;
and (3) probe: 0.8 μ l;
cDNA：1μl；
ddH2o: make up to 20 μ l;
reaction conditions are as follows: 30s at 95 ℃ for 1 cycle; 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles.
As a further improvement of the present invention, the reverse transcription comprises: a genomic DNA removal step and a cDNA synthesis step; wherein, the reaction system in the genome DNA removing step is as follows:
5x gDNA Wiper Mix：2μl；
Template RNA：50ng；
enzyme-free sterile water (RNase-free water): make up to 10. mu.l.
The reaction conditions were as follows: 42 ℃ for 2 min; infinity at 4 ℃;
the reaction system in the cDNA synthesis step is as follows:
reverse transcription primer (10 um): 1 mul;
mixed liquor of the last step: 10 mu l of the mixture;
enzyme-free sterile water (RNase-free water): 5 mu l of the solution;
10x RT Mix：2μl；
HiScript Ⅱ Enzyme Mix：2μl；
the reaction conditions were as follows: 5min at 25 ℃; 50 ℃ for 15 min; 5min at 85 ℃; 4 ℃ and infinity. The reverse transcription product, i.e., cDNA, is obtained.
As a further improvement of the invention, the expression quantity R of the nucleic acid marker is the ratio of the Ct value of the target gene to the Ct value of the reference gene.
As a further improvement of the invention, when the expression level R of the nucleic acid marker satisfies at least three of the following conditions, the Kawasaki disease is judged to be positive; judging that the expression level R of the nucleic acid marker is uncertain when the expression level R of the nucleic acid marker satisfies the following two conditions; when the expression level R of the nucleic acid marker satisfies the following two or less, the result is judged to be negative;
when the target gene is miR-125a-5p, R125a is less than or equal to 1.2;
when the target gene is hsa-miR-3182, R3182 is less than 1;
when the target gene is hsa-miR-3675-5p, R3675 is less than 1.3;
when the target gene is miR-4433b-5p, R4433b is less than or equal to 1.
The invention has the following beneficial effects: platelets are more readily available and high in platelets compared to platelet exosomes. Platelet miRNA is extracted by collecting platelets in blood, reverse transcription is carried out on the miRNA, Ct values of a target gene and an internal reference gene are detected by RT-qPCR, and corresponding results are obtained by calculation. The method can timely and accurately distinguish the children patients with Kawasaki disease from other febrile diseases (such as measles, heat rash, gastroenteritis, hand-foot-mouth disease, herpes and the like), and improves the detection rate of early diagnosis of the Kawasaki disease. Therefore, the kit has great clinical application value for rapid diagnosis of the children with Kawasaki disease, and provides technical support for further developing a rapid diagnosis kit used for the children with Kawasaki disease.
The miRNA in the platelets is collected, and the platelets are easier to obtain and rich in content compared with exosomes, and the extraction method is simple. The invention sets specific miRNA reference genes, and the method is more reasonable. The 4 miRNAs selected by the invention are from the results of second-generation high-depth sequencing, and are obtained by combining clinical information comprehensive analysis scoring, the 4 miRNAs are analyzed in a combined manner, the accuracy is far higher than that of the detection of a single biological standard substance, and the occurrence of false positive is reduced. The Kawasaki disease and the common fever are more easily distinguished. The method has the advantages of convenient material acquisition, low cost, high sensitivity, good stability, easy operation and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a ROC curve for the m125a nucleic acid marker;
FIG. 2 is a ROC curve for the m4433b nucleic acid marker;
FIG. 3 is a ROC curve for the m3182 nucleic acid marker;
FIG. 4 is a ROC curve for the m3675 nucleic acid marker;
FIG. 5 is a ROC curve for the joint analysis of four nucleic acid markers.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
miRNA: MicroRNA, a non-coding single-stranded RNA molecule of about 20-30 nucleotides in length encoded by an endogenous gene, is involved in the regulation of post-transcriptional gene expression in animals and plants.
Example 1 nucleic acid sequence combinations for Kawasaki disease detection
Comprises reverse transcription primers, amplification primers and probe sequences.
The kawasaki disease detection samples are as follows: human blood platelets.
The Kawasaki disease detection markers are as follows: miR-125a-5p, hsa-miR-3182, hsa-miR-3675-5p, miR-4433b-5p and miR-126, wherein miR-126 is used as an internal reference marker.
The reverse transcription primer sequences are as follows:
miR-125a-5p-sl：CAGGGCATCAGCCTGAACCCTGAACCCTGAATAACCTG AAACTGATGCCCTGTCACAG；
hsa-miR-3182-sl：ACGGGAAACTGAGTTCAGTTCCCAGCGTCCATATCAGT TCCCAGTTTCCCGTGACTAC；
hsa-miR-3675-5p-sl：CAGGGCATCAGCCTGAACCCTGAACCCTGAATAAC CTGAAACTGATGCCCTGGAAATC；
miR-4433b-5p-sl：CTTGATGGCTGCCCGTCAAGCCAGTCAAGTATCCAGT CAAGTCAGCCATCAAGACAGGA；
miR-126-sl：TTAGGGTCAGGCCTGAACCCTGAACCCTGAATGCGAACTG ACCTGACCCTAACGCATT；
the amplification primer sequences are as follows:
a forward primer:
miR-125a-5p-F：CCTGAACCCTGAACCCTGA；
hsa-miR-3182-F：AGTTCAGTTCCCAGCGTCC；
hsa-miR-3675-5p-F：CCTGAACCCTGAACCCTGA；
miR-4433b-5p-F：CCCGTCAAGCCAGTCAAGT；
miR-126-F：CCTGAACCCTGAACCCTGA；
reverse primer:
miR-125a-5p-R：GGTCCCTGAGACCCTTTAAC；
hsa-miR-3182-R：TGCGCGCTTCTGTAGT；
hsa-miR-3675-5p-R：CCTCTATGGGGCTTCTGTAGA；
miR-4433b-5p-R：GATGTCCCACCCCCAC；
miR-126-R：GAGCTCGTACCGTGAGTAAT；
the probe sequence is as follows:
miR-125a-5p-P：AACCTGAAACTGATGCCCTG；
hsa-miR-3182-P：ATCAGTTCCCAGTTTCCCGT；
hsa-miR-3675-5p-P：AACCTGAAACTGATGCCCTG；
miR-4433b-5p-P：CCAGTCAAGTCAGCCATCAAG；
miR-126-P：GCGAACTGACCTGACCCTAA。
the probe nucleotide sequence is characterized in that the 5 'end of the probe nucleotide sequence is labeled with a fluorescence reporter group, the 3' end of the probe nucleotide sequence is labeled with a fluorescence quenching group, the fluorescence reporter group of the probe nucleotide sequence is at least one of FAM, HEX, VIC, ROX and Cy5, preferably FAM and HEX, and the fluorescence quenching group is at least one of BHQ1, BHQ2 and MGB, preferably BHQ 1.
Example 2A kit for Kawasaki disease detection
Including reverse transcription reagents, amplification reagents, and probe sequences.
The probe sequence comprises miR-125a-5P-P, hsa-miR-3182-P, hsa-miR-3675-5P-P, miR-4433b-5P-P, miR-126-P; the probe nucleotide sequence is characterized in that a fluorescence reporter group is marked at the 5 'end and a fluorescence quenching group is marked at the 3' end, the fluorescence reporter group of the probe nucleotide sequence is at least one of FAM, HEX, VIC, ROX and Cy5, and the fluorescence quenching group is at least one of BHQ1, BHQ2 and MGB.
The reverse transcription primer sequence comprises miR-125a-5p-sl, hsa-miR-3182-sl, hsa-miR-3675-5p-sl, miR-4433b-5p-sl and miR-126-sl; the amplification primers are a forward primer and a reverse primer; the forward primer comprises miR-125a-5p-F, hsa-miR-3182-F, hsa-miR-3675-5p-F, miR-4433b-5p-F, miR-126-F; the reverse primer comprises miR-125a-5p-R, hsa-miR-3182-R, hsa-miR-3675-5p-R, miR-4433b-5p-R, miR-126-R.
Example 3 detection of Kawasaki disease
(1) The extraction method of platelet miRNA comprises the following steps: the platelet miRNA is extracted by an extraction kit, wherein the extraction kit is a life brand mirVana miRNA Isolation kit (cat # AM 1561). The method comprises the following specific steps:
1) the platelet samples to be extracted were removed from the-80 ℃ freezer, and the cells suspended in cold PBS solution were slowly aspirated using a pipette gun and then placed on ice.
2) Add 300ul Lysis/Binding Buffer to the centrifuge tube, vortex for 30s until no precipitate is present, and centrifuge instantaneously.
3) 30ul of miRNA Homogenate Additive was added to the centrifuge tubes, vortexed, mixed well, centrifuged instantaneously, and incubated on ice for 10 min.
4) To the centrifuge tube was added 300ul of phenol-chloroform-isoamyl alcohol mixture (25: 24: 1) vortex and mix well.
5) Centrifugation was carried out at 10000 Xg for 5min at 4 ℃.
6) The supernatant was removed and transferred to a new 1.5ml centrifuge tube, 375ul 100% ethanol was added, the mixture was inverted and mixed, and the mixture was centrifuged instantaneously.
7) The solution was transferred to a centrifugation column and centrifuged at 10000 Xg for 15s, and the liquid in the collection column was discarded.
8) 700ul of Wash Solution 1 was added to the spin column, centrifuged at 10000 Xg for 15s, and the liquid collected in the column was discarded.
9) 500ul of Wash Solution 2/3 was added to the spin column, and the column was centrifuged at 10000 Xg for 15s, and the liquid collected was discarded.
10) Idling at 10000 Xg for 2min, and air drying at room temperature for 2 min.
11) Transferring the collection column to a new 1.5ml centrifuge tube, adding 100ul of 95 ℃ Solution, incubating for 2min, and centrifuging at 10000 Xg for 2min to obtain platelet miRNA.
(2) Reverse transcription:
adding reverse transcription primers (miR-125 a-5p-sl, hsa-miR-3182-sl, hsa-miR-3675-5p-sl, miR-4433b-5p-sl and miR-126-sl) for reverse transcription to obtain cDNA;
1) genomic DNA removal:
reaction system:
5x gDNA Wiper Mix：2μl；
Template RNA：50ng；
enzyme-free sterile water (RNase-free water): make up to 10. mu.l.
Reaction conditions are as follows: 42 ℃ for 2 min; 4 ℃ and infinity.
2) cDNA Synthesis
Reaction system:
reverse transcription primer (10 μm): 1 mul;
mixed liquor of the last step: 10 mu l of the mixture;
enzyme-free sterile water (RNase-free water): 5 mu l of the solution;
10x RT Mix：2μl；
HiScript Ⅱ Enzyme Mix：2μl；
reaction conditions are as follows: 5min at 25 ℃; 50 ℃ for 15 min; 5min at 85 ℃; 4 ℃ and infinity. The reverse transcription product, i.e., cDNA, is obtained.
(3) And (3) amplification reaction:
and (3) carrying out PCR amplification reaction by using the cDNA as a template and using a forward primer and a reverse primer, and adding a probe sequence while carrying out the amplification reaction.
The amplification reaction system is as follows:
Premix Ex Taq（Probe qPCR）（2X）：10μl；
primer: F/R is 0.4 mu l each;
and (3) probe: 0.8 μ l;
cDNA：1μl；
ddH2o: make up to 20 μ l;
the amplification reaction conditions were as follows: 30s at 95 ℃ for 1 cycle; 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles.
(4) And (4) analyzing results:
and carrying out RT-qPCR analysis on the amplification product, judging the Ct value of the fluorescence quantitative reaction of the miRNA of the target gene and the internal reference gene, and calculating the expression quantity R of the nucleic acid marker and the ratio of the Ct value of the target gene to the Ct value of the internal reference gene.
When the expression level R of the nucleic acid marker meets at least three of the following conditions, the Kawasaki disease can be judged to be positive;
when the expression quantity R of the nucleic acid marker meets the following two conditions, the judgment is uncertain, and retesting is carried out after a certain period is needed;
when the expression level R of the nucleic acid marker satisfies the following two or less, the result is judged to be negative;
when the target gene is miR-125a-5p, R125a is less than or equal to 1.2;
when the target gene is hsa-miR-3182, R3182 is less than 1;
when the target gene is hsa-miR-3675-5p, R3675 is less than 1.3;
when the target gene is miR-4433b-5p, R4433b is less than or equal to 1.
Example 4 specificity test
In the test, 6 normal human samples and 17 Kawasaki patient samples were taken. Platelets of kawasaki disease patients and healthy persons were extracted according to the extraction method in example 3, and miRNA libraries were constructed, subjected to next-generation sequencing, and subjected to bioinformatics analysis. Sequencing result analysis shows that the expression of miR-125a-5p, hsa-miR-3182, hsa-miR-3675-5p and miR-4433b-5p 4 miRNAs in platelets of Kawasaki disease patients is far higher than that of platelets of healthy people (table 1), and nucleic acid marker R values of normal people and Kawasaki disease patients (table 2) are obtained; miR-126 as an internal reference gene is stably expressed in platelets of Kawasaki patients and healthy people.
TABLE 1 comparison of nucleic acid marker R in Normal humans with Kawasaki disease patients
TABLE 2 nucleic acid marker R values of Normal human and Kawasaki disease patients
| Doctor Diagnosis of | miR-125a-5p and intein Reference ratio (R125 a) | miR-4433b-5p and internal reference Object ratio value (R4433 b) | hsa-miR-3182 and Netherlands Reference ratio (R3182) | hsa-miR-3675-5p and endo Reference ratio (R3675) | |
| Cases of disease 1 | Kawasaki (Kawasaki) Negative of | 1.2122 | 1.3261 | 0.9306 | 1.2775 |
| Cases of disease 2 | Kawasaki (Kawasaki) Negative of | 1.2969 | 0.9289 | 1.0663 | 1.3256 |
| Cases of disease 3 | Kawasaki (Kawasaki) Negative of | 1.4192 | 1.0145 | 1.0062 | 1.3790 |
| Cases of disease 4 | Kawasaki (Kawasaki) Negative of | 1.3910 | 0.8874 | 0.9807 | 1.3487 |
| Cases of disease 5 | Kawasaki (Kawasaki) Negative of | 1.4518 | 0.9267 | 0.9407 | 1.3558 |
| Cases of disease 6 | Kawasaki (Kawasaki) Negative of | 1.7104 | 0.9948 | 1.0597 | 1.2801 |
| Cases of disease 7 | Kawasaki (Kawasaki) Positive for | 1.0139 | 0.9582 | 0.8033 | 1.1251 |
| Cases of disease 8 | Kawasaki (Kawasaki) Positive for | 1.2692 | 0.8083 | 0.8542 | 0.6376 |
| Cases of disease 9 | Kawasaki (Kawasaki) Positive for | 1.6996 | 0.9730 | 1.0403 | 0.6816 |
| Cases of disease 10 | Kawasaki (Kawasaki) Positive for | 0.9573 | 0.9330 | 0.7727 | 1.0434 |
| Cases of disease 11 | Kawasaki (Kawasaki) Positive for | 0.9832 | 0.9358 | 0.7682 | 1.0367 |
| Cases of disease 12 | Kawasaki (Kawasaki) Positive for | 1.0249 | 0.9543 | 0.7881 | 1.1045 |
| Cases of disease 13 | Kawasaki (Kawasaki) Positive for | 0.9578 | 0.9512 | 1.1445 | 1.2076 |
| Cases of disease 14 | Kawasaki (Kawasaki) Positive for | 0.9298 | 0.9169 | 1.0788 | 1.1656 |
| Cases of disease 15 | Kawasaki (Kawasaki) Positive for | 1.3355 | 0.9932 | 0.9796 | 1.4960 |
| Cases of disease 16 | Kawasaki (Kawasaki) Positive for | 1.2967 | 0.9499 | 0.9691 | 0.4306 |
| Cases of disease 17 | Kawasaki (Kawasaki) Positive for | 1.3076 | 0.8465 | 0.9079 | 1.2913 |
| Cases of disease 18 | Kawasaki (Kawasaki) Positive for | 1.3822 | 0.8440 | 0.7756 | 1.1684 |
| Cases of disease 19 | Kawasaki (Kawasaki) Positive for | 1.3902 | 0.8227 | 0.8865 | 1.1466 |
| Cases of disease 20 | Kawasaki (Kawasaki) Positive for | 1.0392 | 0.9691 | 1.1036 | 1.2097 |
| Cases of disease 21 | Kawasaki (Kawasaki) Positive for | 1.0130 | 1.0135 | 1.2283 | 1.2757 |
| Cases of disease 22 | Kawasaki (Kawasaki) Positive for | 0.9143 | 0.9103 | 1.0710 | 1.1583 |
| Cases of disease 23 | Kawasaki (Kawasaki) Positive for | 1.4259 | 0.8180 | 0.8243 | 1.1314 |
TABLE 3 results of normal and Kawasaki disease patients
| Doctor Diagnosis of | miR-125a-5p and internal reference Ratio of things (R) | miR-4433b-5p and internal Reference ratio (R) | hsa-miR-3182 and Netherlands Reference ratio (R) | hsa-miR-3675-5p and endo Reference ratio (R) | Judgment kit Broken result | |
| Disease and illness Example 1 | Kawasaki (Kawasaki) Negative of | Negative of | Negative of | Positive for | Positive for | Uncertainty |
| Disease and illness Example 2 | Kawasaki (Kawasaki) Negative of | Negative of | Positive for | Negative of | Negative of | Negative of |
| Disease and illness Example 3 | Kawasaki (Kawasaki) Negative of | Negative of | Negative of | Negative of | Negative of | Negative of |
| Disease and illness Example 4 | Kawasaki (Kawasaki) Negative of | Negative of | Positive for | Positive for | Negative of | Uncertainty |
| Disease and illness Example 5 | Kawasaki (Kawasaki) Negative of | Negative of | Positive for | Positive for | Negative of | Uncertainty |
| Disease and illness Example 6 | Kawasaki (Kawasaki) Negative of | Negative of | Positive for | Negative of | Positive for | Uncertainty |
| Disease and illness Example 7 | Kawasaki (Kawasaki) Positive for | Positive for | Positive for | Positive for | Positive for | Positive for |
| Disease and illness Example 8 | Kawasaki (Kawasaki) Positive for | Negative of | Positive for | Positive for | Positive for | Positive for |
| Disease and illness Example 9 | Kawasaki (Kawasaki) Positive for | Negative of | Positive for | Negative of | Positive for | Uncertainty |
| Disease and illness Example (b) 10 | Kawasaki (Kawasaki) Positive for | Positive for | Positive for | Positive for | Positive for | Positive for |
| Disease and illness Example (b) 11 | Kawasaki (Kawasaki) Positive for | Positive for | Positive for | Positive for | Positive for | Positive for |
| Disease and illness Example (b) 12 | Kawasaki (Kawasaki) Positive for | Positive for | Positive for | Positive for | Positive for | Positive for |
| Disease and illness Example (b) 13 | Kawasaki (Kawasaki) Positive for | Positive for | Positive for | Negative of | Positive for | Positive for |
| Disease and illness Example (b) 14 | Kawasaki (Kawasaki) Positive for | Positive for | Positive for | Negative of | Positive for | Positive for |
| Disease and illness Example (b) 15 | Kawasaki (Kawasaki) Positive for | Negative of | Positive for | Positive for | Negative of | Uncertainty |
| Disease and illness Example (b) 16 | Kawasaki (Kawasaki) Positive for | Negative of | Positive for | Positive for | Positive for | Positive for |
| Disease and illness Example (b) 17 | Kawasaki (Kawasaki) Positive for | Negative of | Positive for | Positive for | Positive for | Positive for |
| Disease and illness Example (b) 18 | Kawasaki (Kawasaki) Positive for | Negative of | Positive for | Positive for | Positive for | Positive for |
| Disease and illness Example (b) 19 | Kawasaki (Kawasaki) Positive for | Negative of | Positive for | Positive for | Positive for | Positive for |
| Disease and illness Example (b) 20 | Kawasaki (Kawasaki) Positive for | Positive for | Positive for | Negative of | Positive for | Positive for |
| Disease and illness Example (b) 21 | Kawasaki (Kawasaki) Positive for | Positive for | Negative of | Negative of | Positive for | Uncertainty |
| Disease and illness Example (b) 22 | Kawasaki (Kawasaki) Positive for | Positive for | Positive for | Negative of | Positive for | Positive for |
| Disease and illness Example (b) 23 | Kawasaki (Kawasaki) Positive for | Negative of | Positive for | Positive for | Positive for | Positive for |
Results of 23 ROC curve analyses:
sensitivity and specificity are obtained by taking m125a, m4433b, m3182 and m3675 nucleic acid markers as judgment standards and counting true positive, false positive, true negative and false negative respectively. The results are shown in Table 4. In the ROC curves (fig. 1 to 5), the areas under the ROC curves are 0.8137, 0.6764, 0.6274, 0.9215 and 0.9411, respectively, indicating good judgment results. The ROC curve is a characteristic curve of the operation of a subject, the ordinate (sensitivity) is a true positive rate, and the abscissa (1-specificity) is a false positive rate. The AUC value of the ROC curve represents the area under the curve, generally between 0.5 and 1, with larger values representing better classification of the curve. The AUC (area under the curve) of the combined analysis of the four RNAs was 0.9411, indicating a good classification effect for healthy and kawasaki patients. In the combined analysis of the four nucleic acid markers, when the R values of three or more nucleic acid markers meet the judgment standard, the sample is considered to be positive for Kawasaki disease to a great extent. The results show that the sensitivity, specificity and accuracy of the four nucleic acid marker assays are 100.00%, 66.67% and 86.96%, indicating that the detection method has good detection capability.
TABLE 4 nucleic acid marker sensitivity and specificity
| Nucleic acid markers | m125a | m4433b | m3182 | m3675 | Combined analysis of four markers |
| Sensitivity of the probe | 100.00% | 80.00% | 82.35% | 90.00% | 100.00% |
| Degree of specificity | 42.86% | 66.67% | 25.00% | 44.44% | 66.67% |
| Accuracy of | 65.22% | 78.26% | 73.91% | 95.65% | 86.96% |
Compared with the prior art, the platelet is easier to obtain and has high platelet content compared with platelet exosomes. Platelet miRNA is extracted by collecting platelets in blood, reverse transcription is carried out on the miRNA, Ct values of a target gene and an internal reference gene are detected by RT-qPCR, and corresponding results are obtained by calculation. The method can timely and accurately distinguish the children patients with Kawasaki disease from other febrile diseases (such as measles, heat rash, gastroenteritis, hand-foot-mouth disease, herpes and the like), and improves the detection rate of early diagnosis of the Kawasaki disease. Therefore, the kit has great clinical application value for rapid diagnosis of the children with Kawasaki disease, and provides technical support for further developing a rapid diagnosis kit used for the children with Kawasaki disease.
The miRNA in the platelets is collected, and the platelets are easier to obtain and rich in content compared with exosomes, and the extraction method is simple. The invention sets specific miRNA reference genes, and the method is more reasonable. The 4 miRNAs selected by the invention are from the results of second-generation high-depth sequencing, and are obtained by combining clinical information comprehensive analysis scoring, the 4 miRNAs are analyzed in a combined manner, the accuracy is far higher than that of the detection of a single biological standard substance, and the occurrence of false positive is reduced. The Kawasaki disease and the common fever are more easily distinguished. The method has the advantages of convenient material acquisition, low cost, high sensitivity, good stability, easy operation and the like.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Dao precision medical science and technology (Shanghai) Co., Ltd
<120> primer group, probe and kit for detecting Kawasaki disease
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Claims (10)
1. A nucleic acid sequence combination for Kawasaki disease detection comprises a reverse transcription primer, an amplification primer and a probe sequence, and is characterized in that the nucleic acid sequence combination can effectively detect miR-125a-5p, hsa-miR-3182, hsa-miR-3675-5p, miR-4433b-5p and miR-126 in human blood platelets qualitatively/quantitatively, wherein miR-126 serves as an internal reference marker;
wherein, the reverse transcription primer sequence is as follows:
miR-125a-5p-sl：CAGGGCATCAGCCTGAACCCTGAACCCTGAATAACCT GAAACTGATGCCCTGTCACAG；
hsa-miR-3182-sl：ACGGGAAACTGAGTTCAGTTCCCAGCGTCCATATCAG TTCCCAGTTTCCCGTGACTAC；
hsa-miR-3675-5p-sl：CAGGGCATCAGCCTGAACCCTGAACCCTGAATAAC CTGAAACTGATGCCCTGGAAATC；
miR-4433b-5p-sl：CTTGATGGCTGCCCGTCAAGCCAGTCAAGTATCCAGT CAAGTCAGCCATCAAGACAGGA；
miR-126-sl：TTAGGGTCAGGCCTGAACCCTGAACCCTGAATGCGAACTG ACCTGACCCTAACGCATT；
the amplification primer sequences are as follows:
a forward primer:
miR-125a-5p-F：CCTGAACCCTGAACCCTGA；
hsa-miR-3182-F：AGTTCAGTTCCCAGCGTCC；
hsa-miR-3675-5p-F：CCTGAACCCTGAACCCTGA；
miR-4433b-5p-F：CCCGTCAAGCCAGTCAAGT；
miR-126-F：CCTGAACCCTGAACCCTGA；
reverse primer:
miR-125a-5p-R：GGTCCCTGAGACCCTTTAAC；
hsa-miR-3182-R：TGCGCGCTTCTGTAGT；
hsa-miR-3675-5p-R：CCTCTATGGGGCTTCTGTAGA；
miR-4433b-5p-R：GATGTCCCACCCCCAC；
miR-126-R：GAGCTCGTACCGTGAGTAAT；
the probe sequence is as follows:
miR-125a-5p-P：AACCTGAAACTGATGCCCTG；
hsa-miR-3182-P：ATCAGTTCCCAGTTTCCCGT；
hsa-miR-3675-5p-P：AACCTGAAACTGATGCCCTG；
miR-4433b-5p-P：CCAGTCAAGTCAGCCATCAAG；
miR-126-P：GCGAACTGACCTGACCCTAA。
2. a kit for detecting kawasaki disease, comprising a reverse transcription reagent, an amplification reagent and the probe sequence of claim 1, wherein the 5 'end of the probe nucleotide sequence is labeled with a fluorescence reporter group, the 3' end of the probe nucleotide sequence is labeled with a fluorescence quenching group, the fluorescence reporter group of the probe nucleotide sequence is at least one of FAM, HEX, VIC, ROX and Cy5, and the fluorescence quenching group is at least one of BHQ1, BHQ2 and MGB; the reverse transcription primer sequence is the reverse transcription primer of claim 1; the amplification primers are the forward primer and the reverse primer of claim 1.
3. A detection chip or device comprising the reverse transcription primer, amplification primer and probe sequence of claim 1.
4. Use of the reverse transcription primers, amplification primers and probe sequences of claim 1 in the preparation of reagents or tools for prediction of Kawasaki disease, and for auxiliary diagnosis of Kawasaki disease.
5. Use of the nucleic acid sequence combination of claim 1 for the preparation of reagents for qualitative and/or quantitative detection of miR-125a-5p, hsa-miR-3182, hsa-miR-3675-5p, miR-4433b-5p and miR-126.
6. A method for detecting target genes miR-125a-5p, hsa-miR-3182, hsa-miR-3675-5p, miR-4433b-5p and reference genes miR-126 comprises the following steps:
1) extracting platelet miRNA from a sample, adding reverse transcription primers miR-125a-5p-sl, hsa-miR-3182-sl, hsa-miR-3675-5p-sl, miR-4433b-5p-sl and miR-126-sl for reverse transcription to obtain cDNA;
2) using cDNA as a template, performing amplification reaction by using a forward primer and a reverse primer, adding a probe sequence during the amplification reaction, performing RT-qPCR analysis on an amplification product, judging the Ct value of the fluorescence quantitative reaction of miRNA of a target gene and an internal reference gene, and calculating the expression quantity R of a nucleic acid marker;
the forward primer comprises miR-125a-5p-F, hsa-miR-3182-F, hsa-miR-3675-5p-F, miR-4433b-5p-F, miR-126-F;
the reverse primer comprises miR-125a-5p-R, hsa-miR-3182-R, hsa-miR-3675-5p-R, miR-4433b-5p-R, miR-126-R;
the probe sequence comprises miR-125a-5P-P, hsa-miR-3182-P, hsa-miR-3675-5P-P, miR-4433b-5P-P, miR-126-P;
wherein the reverse transcription primers, amplification primers and probe sequences are as described in claim 1.
7. The method of claim 6, wherein the amplification reaction system and conditions are as follows:
Premix Ex Taq（Probe qPCR）（2X）：10μl；
primer: F/R is 0.4 mu l each;
and (3) probe: 0.8 μ l;
cDNA：1μl；
ddH2o: make up to 20 μ l;
reaction conditions are as follows: 30s at 95 ℃ for 1 cycle; 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles.
8. The method of claim 6, wherein the reverse transcription comprises: a genomic DNA removal step and a cDNA synthesis step;
wherein, the reaction system in the genome DNA removing step is as follows:
5x gDNA Wiper Mix：2μl；
Template RNA：50ng；
RNase-free water: make up to 10. mu.l.
The reaction conditions were as follows: 42 ℃ for 2 min; infinity at 4 ℃;
the reaction system in the cDNA synthesis step is as follows:
reverse transcription primer (10 μm): 1 mul;
mixed liquor of the last step: 10 mu l of the mixture;
RNase-free water：5μl；
10x RT Mix：2μl；
HiScript Ⅱ Enzyme Mix：2μl；
the reaction conditions were as follows: 5min at 25 ℃; 50 ℃ for 15 min; 5min at 85 ℃; 4 ℃ and infinity. The reverse transcription product, i.e., cDNA, is obtained.
9. The expression quantity R of the nucleic acid marker is the ratio of the Ct value of the target gene to the Ct value of the reference gene.
10. The method according to claim 9, wherein the Kawasaki disease is determined to be positive when the expression level R of the nucleic acid marker satisfies at least three of the following conditions; judging that the expression level R of the nucleic acid marker is uncertain when the expression level R of the nucleic acid marker satisfies the following two conditions; when the expression level R of the nucleic acid marker satisfies the following two or less, the result is judged to be negative;
when the target gene is miR-125a-5p, R125a is less than or equal to 1.2;
when the target gene is hsa-miR-3182, R3182 is less than 1;
when the target gene is hsa-miR-3675-5p, R3675 is less than 1.3;
when the target gene is miR-4433b-5p, R4433b is less than or equal to 1.
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Application publication date: 20220405 Assignee: Shanghai Haoen Medical Technology Co.,Ltd. Assignor: Daozhi precision medicine technology (Shanghai) Co.,Ltd. Contract record no.: X2024980005551 Denomination of invention: Primer sets, probes, and test kits for detecting Kawasaki disease Granted publication date: 20231205 License type: Open License Record date: 20240510 |