CN116426634A - Gene detection reagent and kit for cornea malnutrition - Google Patents

Gene detection reagent and kit for cornea malnutrition Download PDF

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CN116426634A
CN116426634A CN202310444713.6A CN202310444713A CN116426634A CN 116426634 A CN116426634 A CN 116426634A CN 202310444713 A CN202310444713 A CN 202310444713A CN 116426634 A CN116426634 A CN 116426634A
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cornea
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于晓光
吴文辉
李小玲
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Wenzhou Puxi Gene Technology Co ltd
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Abstract

The invention provides a gene detection kit for cornea malnutrition, which comprises a blood sample pretreatment reagent and an amplification reagent, wherein the blood sample pretreatment reagent comprises the following components: 1-50mM Tris,1-20mM guanidine iso-sulfate, 20-80mM lauryl alcohol, 300-500mM erythritol, 5000-8000U/mL neutral proteinase, the balance being water, and the pH value is 6.5-7.5; the amplification reagent comprises amplification primers and probes. When the kit provided by the invention detects cornea dystrophy from a whole blood sample, the operation is simple and convenient, the time consumption is short, no additional nucleic acid extraction operation is needed, the detection accuracy of the kit is high, and the kit can be used for diagnosis of the cause of cornea dystrophy and gene screening, and has a wide clinical application prospect.

Description

Gene detection reagent and kit for cornea malnutrition
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a gene detection reagent and a kit for cornea dystrophy.
Background
Corneal dystrophy is a generic term for a series of primary progressive keratopathy related to familial inheritance. Most of the diseases are inherited by autosomal dominant; the cornea is primarily affected by the cornea, so that the eyes are symmetrical; slow disease course, and no new blood vessel growth in lesion area; starting to violate only a certain layer of the cornea; late stage can reach adjacent layers and even affect the full-thickness cornea; the drug treatment is ineffective. The cornea transplantation operation treatment can be performed on the patients with the influence of vision.
The early gene screening for cornea malnutrition is beneficial to early intervention and avoids the situation of aggravation of later-stage illness. Mutation sites on some genes related to corneal dystrophy, such as tgfβi gene, CHST6 gene, big 3 gene, TMCO3 gene, etc., have been disclosed in the prior art, and primer and probe designs are mostly performed for the mutation sites on these genes, PCR amplification is performed by extracting DNA, and diagnosis of corneal dystrophy is performed after sequencing.
Chinese patent CN201711147057.4 discloses a primer probe for diagnosing corneal dystrophy caused by human tgfβi gene 124 site variation, comprising specific primers designed for tgfβi gene 124 site: a specific wild-type Taqman fluorescent probe for wild-type site R124, and at least one of four specific mutant Taqman fluorescent probes for mutant sites C124, S124, H124, and L124, each probe being linked to a different fluorescent reporter group for separate monitoring by collecting different fluorescent signals, and without the need for adding an internal reference. The method for detecting the human TGF-beta I gene 124 locus variation based on the primer probe has the advantages of high sensitivity, strong specificity, difficult pollution, good accuracy, convenience, rapidness and the like, and is suitable for being applied to clinical case analysis and inspection work.
Chinese patent CN201611138588.2 discloses a kit for detecting mutation sites of granular cornea dystrophy susceptibility genes. The kit comprises a primer pair A, a primer pair B, a primer pair C, an extension primer A1, an extension primer A2, an extension primer B and an extension primer C; the kit can detect the nucleotides of rs121909208, rs121909209, rs121909211 and rs121909215, and can also be used for auxiliary diagnosis of granular cornea malnutrition.
However, in the prior art, the gene diagnosis of the cornea malnutrition is carried out by DNA extraction and then amplification, the operation is complicated and long-time consuming, and the development of a kit with higher detection efficiency is necessary for clinic.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a gene detection kit which can directly apply a blood sample to cornea malnutrition, simultaneously amplify a plurality of relevant sites, has short time consumption and is easy to operate, so as to perform detection more quickly and accurately.
In one aspect, the invention provides a gene detection kit for corneal dystrophy.
Specifically, the kit comprises a blood sample pretreatment reagent and an amplification reagent.
Further specifically, the blood sample pretreatment reagent includes: 1-50mM Tris,1-20mM guanidine isocyanate, 20-80mM lauryl alcohol, 300-500mM erythritol, 5000-8000U/mL neutral protease, and water as the rest, wherein the pH is 6.5-7.5; the amplification reagent comprises amplification primers and probes.
Preferably, the pretreatment reagent includes: 20-40mM Tris,10-20mM guanidine isocyanate, 20-60mM lauryl alcohol, 400-500mM erythritol and 5000-6000U/mL neutral protease.
Further preferably, the pretreatment reagent includes: 25-40mM Tris,10-15mM guanidine isocyanate, 50-60mM lauryl alcohol, 400-500mM erythritol and 5000-6000U/mL neutral protease.
Still further, the pretreatment reagent includes: 25mM Tris,10mM guanidine isocyanate, 50mM lauryl alcohol, 500mM erythritol and 5000U/mL neutral protease.
The kit can be directly used for blood sample amplification detection. The blood sample may be plasma or serum.
Specifically, the amplification primers and probes are used for detecting TGF beta I gene mutation: c.370C > T (rs 121909210), c.371G > T/A (rs 121909211), c.1663C > T (rs 121909208) and c.1664G > A (rs 121909209).
Preferably, the amplification primers and probes are as follows:
Figure BDA0004195160530000021
the probe sequence markers were as follows:
SEQ ID NO.5: a VIC is marked at the 5 'end, and an MGB is marked at the 3' end;
SEQ ID NO.6:5 'end marks FAM and 3' end marks MGB;
SEQ ID NO.7: a VIC is marked at the 5 'end, and an MGB is marked at the 3' end;
SEQ ID NO.8:5 'end marks FAM and 3' end marks MGB;
SEQ ID NO.9: a ROX is marked at the 5 'end, and an MGB is marked at the 3' end;
SEQ ID NO.10: a VIC is marked at the 5 'end, and an MGB is marked at the 3' end;
SEQ ID NO.11:5 'end marks FAM and 3' end marks MGB;
SEQ ID NO.12: the 5 'end marks FAM and the 3' end marks MGB.
The primer or probe is used at a concentration of 0.5 to 1.5. Mu.M.
The kit also comprises a PCR amplification buffer system, wherein the buffer system comprises: 100mM Tris-HCl,10-15mM MgCl 2 20-30mM KCl,20-50mM glacial acetic acid.
Preferably, the buffer system comprises: 100mM Tris-HCl,12-14mM MgCl 2 20-25mM KCl,20-30mM glacial acetic acid.
Further preferably, the buffer system comprises: 100mM Tris-HCl,14mM MgCl 2 22mM KCl,25mM glacial acetic acid.
The kit may also include other reagents for PCR amplification.
Preferably, the amplification system for which the kit is suitable is as follows:
reagent(s) Dosage (mu L)
PCR amplification buffer system (10×) 2.5
DNA polymerase (5U/. Mu.L) 1
dNTPs(2.5mM each) 2
Equivalent mixture of primer and probe 3(3.5)
Template 5
ddH 2 O Complement 25
The amplification procedure that can be used for the kit is: 5min at 95 ℃ (fluorescence was collected at 95 ℃ for 10s and 60 ℃ for 30 s) 40cycles.
The kit can also comprise a positive quality control product and a negative quality control product.
In another aspect, the present invention provides a blood sample pretreatment reagent.
Specifically, the blood sample pretreatment reagent comprises: 1-50mM Tris,1-20mM guanidine isocyanate, 20-80mM lauryl alcohol, 300-500mM erythritol, 5000-8000U/mL neutral protease, and water as the rest, wherein the pH is 6.5-7.5.
The pretreatment reagent for the blood sample is directly added into the blood sample for use.
Preferably, the blood sample pretreatment reagent comprises: 20-40mM Tris,10-20mM guanidine isocyanate, 20-60mM lauryl alcohol, 400-500mM erythritol and 5000-6000U/mL neutral protease.
Further preferably, the pretreatment reagent includes: 25-40mM Tris,10-15mM guanidine isocyanate, 50-60mM lauryl alcohol, 400-500mM erythritol and 5000-6000U/mL neutral protease.
Still further, the pretreatment reagent includes: 25mM Tris,10mM guanidine isocyanate, 50mM lauryl alcohol, 500mM erythritol and 5000U/mL neutral protease.
In yet another aspect, the invention provides a mixture of amplification primers and probes for use in detecting corneal dystrophies.
The mixture is used for amplifying TGF beta I gene mutation: c.370C > T (rs 121909210), c.371G > T/A (rs 121909211), c.1663C > T (rs 121909208) and c.1664G > A (rs 121909209).
The invention also provides application of the blood sample pretreatment reagent and primer probe mixture in preparing a kit for detecting cornea malnutrition.
The kit is a blood sample detection kit.
Other reagents for PCR amplification may also be included in the kit.
Other reagents for PCR amplification include, but are not limited to: DNA polymerase, positive quality control, negative quality control, dNTPs, etc.
The invention has the beneficial effects that:
when the kit provided by the invention detects cornea dystrophy from a whole blood sample, the operation is simple and convenient, the time consumption is short, no additional nucleic acid extraction operation is needed, the detection accuracy of the kit is high, and the kit can be used for diagnosis of the cause of cornea dystrophy and gene screening, and has a wide clinical application prospect.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
In the following examples:
the enzymes for PCR amplification were Taq DNA polymerase, available from Takara under the trade designation R001A, at a concentration of 5U/. Mu.L.
10 XBuffer was purchased from Takara under the designation R001A.
The positive quality control is recombinant plasmid with positive detection targets, and the solvent is human serum;
the negative quality control product is human serum;
human serum was purchased from Merck under the product number SAE0012.
Neutral protease was purchased from soribao under the accession number Z8031.
Example 1
The kit in this example comprises the following components:
(1) Pretreatment reagent for blood sample: 25mM Tris,10mM guanidine isocyanate, 50mM lauryl alcohol, 500mM erythritol, 5000U/mL neutral protease, and water as the rest; the pH was 7.
(2) PCR amplification buffer: 100mM Tris-HCl,15mM MgCl 2 20mM KCl,30mM glacial acetic acid.
(3) DNA polymerase: 5U/. Mu.L.
(4)dNTPs：2.5mM each。
(5) Primer probe mixture
Figure BDA0004195160530000051
The probe sequence markers were as follows:
SEQ ID NO.5: a VIC is marked at the 5 'end, and an MGB is marked at the 3' end;
SEQ ID NO.6:5 'end marks FAM and 3' end marks MGB;
SEQ ID NO.7: a VIC is marked at the 5 'end, and an MGB is marked at the 3' end;
SEQ ID NO.8:5 'end marks FAM and 3' end marks MGB;
SEQ ID NO.9: a ROX is marked at the 5 'end, and an MGB is marked at the 3' end;
SEQ ID NO.10: a VIC is marked at the 5 'end, and an MGB is marked at the 3' end;
SEQ ID NO.11:5 'end marks FAM and 3' end marks MGB;
SEQ ID NO.12: the 5 'end marks FAM and the 3' end marks MGB.
Each primer had a concentration of 10. Mu.M and each probe had a concentration of 10. Mu.M.
Example 2
The difference from example 1 is that the pretreatment reagent for blood sample is: 40mM Tris,15mM guanidine isocyanate, 60mM lauryl alcohol, 400mM erythritol, neutral protease 6000U/mL, the balance being water; the pH was 7.
Example 3
The difference from example 1 is that the pretreatment reagent for blood sample is: 20mM Tris,20mM guanidine isocyanate, 50mM lauryl alcohol, 400mM erythritol, 5000U/mL neutral protease, and water as the rest; the pH was 7.
Example 4
The difference from example 1 is that the PCR amplification buffer system comprises: 100mM Tris-HCl,10mM MgCl 2 30mM KCl,40mM glacial acetic acid.
Example 5A method of detecting corneal dystrophy
The specific procedure for detecting malnutrition of the present invention is illustrated by the kit of example 1:
(1) Collecting a blood sample, standing for 5-10min, and adding a blood sample pretreatment reagent into the blood sample, wherein the volume ratio of the blood sample to the pretreatment reagent is 2:1, lightly mixing and standing for 5min to be used as a template for PCR amplification.
(2) The PCR amplification system is as follows:
Figure BDA0004195160530000061
Figure BDA0004195160530000071
qPCR was performed in 4 wells.
(3) The PCR amplification procedure was: 5min at 95 ℃ (fluorescence was collected at 95 ℃ for 10s and 60 ℃ for 30 s) 40cycles.
(4) Result determination method:
genotype determination can be performed only when yin-yang quality control meets the following 3 points at the same time.
Negative quality control: the non-exponential increase period or Ct value of each detection target amplification curve is more than 35;
controlling the nature of yang: each detection target amplification curve has an exponential growth period and Ct value less than or equal to 35;
the two items are required to be simultaneously satisfied in the same test, otherwise, the current test is regarded as invalid and needs to be detected again.
Figure BDA0004195160530000072
Experimental example 1 crowd sample detection
20 patients with cornea malnutrition and 20 healthy people are selected to verify the effect of the invention.
The clinical diagnosis results of each patient are as follows:
numbering device Clinical diagnostic results (Condition) Numbering device Clinical diagnostic results (Condition)
1 Reis-Bucker's corneal dystrophy 11 Fuchs corneal endothelial cell dystrophy
2 Avellino corneal dystrophy 12 Avellino corneal dystrophy
3 Avellino corneal dystrophy 13 Lattice type I corneal dystrophy
4 Fuchs corneal endothelial cell dystrophy 14 Avellino corneal dystrophy
5 Malnutrition of the corneal epithelium and pre-elastic layer 15 Thiel-Behnke corneal dystrophy
6 Plaque-like corneal dystrophy 16 Fuchs corneal endothelial cell dystrophy
7 Avellino corneal dystrophy 17 Gel drop-like corneal dystrophy
8 Granular corneal dystrophy type I 18 Granular corneal dystrophy type I
9 Avellino corneal dystrophy 19 Avellino corneal dystrophy
10 Granular corneal dystrophy type I 20 Avellino corneal dystrophy
After blood collection, the test was performed using the kits of examples 1-4, respectively, wherein 5 positive patients (numbers 2, 9, 13, 14, 18) were detected in total and the healthy population was negative.
In order to confirm the effect of the kit, the relevant genes of the patient sample are detected, and no target mutation is found in the sample with no detected positive, so that the kit is effective at least for diagnosing the corneal dystrophy caused by the target mutation.
Experimental example 2 anti-interference capability of kit
The PCR detection is directly carried out by the blood sample, and particularly, the components in the blood sample, particularly the anticoagulant components, need to be added after sampling, can influence the final detection result, so that the anti-interference capability of the detection kit is necessary.
Considering the blood collection amount problem, 5 positive samples in the example 1 are adopted to respectively verify the anti-interference capability on anticoagulants with different concentrations, and two anticoagulants, namely EDTA and heparin, are detected in total; the dosage of EDTA is set to 0.5g/L, 1g/L and 1.5g/L; the heparin dosage was set to 10IU/mL and 12IU/mL.
The specific arrangement is as follows:
Figure BDA0004195160530000081
Figure BDA0004195160530000091
the positive results of the groups can be detected under the condition of containing different anticoagulants, which shows that the kit provided by the invention has better anti-interference capability, has anti-interference capability on EDTA of at least 1.5g/L and has anti-interference capability on heparin of at least 12IU/mL.
The anti-interference capability experiment is carried out on the kits of the examples 1 to 4 through the cationic quality control, and the dosage of EDTA is set to be 0.5g/L, 1g/L, 1.5g/L and 2g/L; the heparin dosage was set to 10IU/mL, 12IU/mL, 14IU/mL. The anti-interference concentration results for EDTA and heparin were as follows:
examples EDTA Heparin
Example 1 2g/L 14IU/mL
Example 2 2g/L 14IU/mL
Example 3 2g/L 14IU/mL
Example 4 2g/L 14IU/mL
Experimental example 3 sensitivity of kit
And detecting the sensitivity of the kit by using a quality control product.
The detection of the quality control products with different concentrations is carried out, and the quality control products with the concentrations of 0.1 ng/. Mu.L, 0.5 ng/. Mu.L, 1 ng/. Mu.L and 2 ng/. Mu.L are respectively set, so that the detection results show that the minimum detection limit of the kit of the embodiment 1-4 of the application is 0.1 ng/. Mu.L.
Comparative example
A comparative example was set up with reference to example 1, specifically as follows:
Figure BDA0004195160530000092
Figure BDA0004195160530000101
referring to the method of experimental example 1, 5 positive samples were detected, and the results were as follows:
comparative example 1: positive mutations were still detectable in 5 samples, but the Ct values for VIC and FAM channels were increased by 1-3.
Comparative example 2: positive mutations were not detected in 3 out of 5 samples.
Comparative example 3: positive mutations were undetectable in 2 out of 5 samples.
Comparative example 4: 1 out of 5 samples failed to detect positive mutations.
Referring to the method of experimental example 2, the anti-interference ability of comparative examples 1 to 4 when samples 2, 9, 13, 14 were examined was as follows:
comparative example EDTA Heparin
Comparative example 1 (test sample 2) 1g/L 10IU/mL
Comparative example 2 (test sample 9) < 0.5g/L (setting range without result) ＜10IU/mL
Comparative example 3 (test sample 13) 1g/L 10IU/mL
Comparative example 4 (test sample 14) 0.5g/L 10IU/mL
Sample 18 was tested with the kit of comparative example 2 for tamper resistance, considering whether it was a sample problem. The results show that the results of comparative example 2 are not detected within the set EDTA and heparin ranges, and the anti-interference concentration of the EDTA of the comparative example 2 is less than 0.5g/L and the anti-interference concentration of the heparin is less than 10IU/mL.
The anti-interference ability of the kits of comparative examples 1 to 4 was tested and the results were as follows:
comparative example EDTA Heparin
Comparative example 1 0.5g/L 12IU/mL
Comparative example 2 2g/L 14IU/mL
Comparative example 3 2g/L 14IU/mL
Comparative example 4 2g/L 14IU/mL

Claims (15)

1. A gene detection kit for corneal dystrophy, comprising a blood sample pretreatment reagent and an amplification reagent, wherein the blood sample pretreatment reagent comprises: 1-50mM Tris,1-20mM guanidine isocyanate, 20-80mM lauryl alcohol, 300-500mM erythritol, 5000-8000U/mL neutral protease, and water as the rest, wherein the pH is 6.5-7.5; the amplification reagent comprises amplification primers and probes.
2. The kit of claim 1, wherein the pretreatment reagent comprises: 20-40mM Tris,10-20mM guanidine isocyanate, 20-60mM lauryl alcohol, 400-500mM erythritol and 5000-6000U/mL neutral protease.
3. The kit of claim 2, wherein the pretreatment reagent comprises: 25mM Tris,10mM guanidine isocyanate, 50mM lauryl alcohol, 500mM erythritol and 5000U/mL neutral protease.
4. A kit according to claim 3, for direct use in a blood sample amplification assay.
5. The kit of claim 1, wherein the amplification primers and probes are used to detect tgfβi gene mutations: c.370C > T (rs 121909210), c.371G > T/A (rs 121909211), c.1663C > T (rs 121909208) and c.1664G > A (rs 121909209).
6. The kit of claim 5, wherein the amplification primer is SEQ ID NO.1-4 and the probe is SEQ ID NO.5-12.
7. The kit of claim 6, further comprising a buffer system for PCR amplification, wherein the buffer system comprises: 100mM Tris-HCl,10-15mM MgCl 2 20-30mM KCl,20-50mM glacial acetic acid.
8. The kit according to claim 7, wherein the buffer system for PCR amplification comprises: 100mM Tris-HCl,14mM MgCl 2 22mM KCl,25mM glacial acetic acid.
9. The kit of claim 8, further comprising other reagents for PCR reactions, such as: DNA polymerase, dNTPs, and quality control.
10. The kit of claim 9, further comprising a positive quality control and a negative quality control.
11. A blood sample pretreatment reagent comprising: 1-50mM Tris,1-20mM guanidine isocyanate, 20-80mM lauryl alcohol, 300-500mM erythritol, 5000-8000U/mL neutral protease, and water as the rest, wherein the pH is 6.5-7.5.
12. The mixture of the amplification primer and the probe for detecting the cornea malnutrition is characterized in that the amplification primer is SEQ ID NO.1-4, and the probe is SEQ ID NO.5-12.
13. Use of the blood sample pretreatment reagent of claim 11 and the mixture of claim 12 for the preparation of a kit for the detection of corneal dystrophies.
14. The kit of claim 13, wherein the kit is a blood sample detection kit.
15. The kit of claim 14, further comprising other reagents for PCR amplification, such as: DNA polymerase, positive quality control, negative quality control, dNTPs.
CN202310444713.6A 2023-04-24 2023-04-24 Gene detection reagent and kit for cornea malnutrition Pending CN116426634A (en)

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