CN118932045A - A SLC10A1 allele typing detection primer and method - Google Patents
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- 201000006346 Neonatal Jaundice Diseases 0.000 description 2
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- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
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Abstract
The invention provides a SLC10A1 allele genotyping detection primer and a method, wherein the genotyping primer comprises an upstream primer and a downstream primer, the upstream primer is shown as SEQ ID NO.1, and the downstream primer is shown as SEQ ID NO. 2. The parting detection primer provided by the invention has good amplifiability on TT type, CC type and CT type of SLC10A1 gene c.800C & gtT locus under the same amplification condition, can effectively improve the resolution of a melting curve in HRM detection, effectively distinguish TT type, CC type and CT type, and can simply, quickly and efficiently realize parting detection of SLC10A1 gene c.800C & gtT locus, and has the advantages of high accuracy, good specificity, good repeatability and low cost.
Description
Technical Field
The invention belongs to the technical field of molecular diagnosis, and particularly relates to a SLC10A1 allele genotyping detection primer and a method.
Background
Sodium taurocholate cotransporter polypeptide (sodium taurocholate cotransporting polypeptide, NTCP) deficiency is a new inherited bile acid metabolism disorder caused by double allelic mutation of SLC10 A1. NTCP deficiency is a major clinical feature of hyperbilirubinemia, which is remarkable and persistent in childhood, and may be involved in the formation of neonatal hyperbilirubinemia, early-stage cholestasis and cholestasis of pregnancy. In the reported NTCP-deficient patients, the SLC10A1 gene mutation c.800C > T (p.Ser267Phe) is most common, the allele frequency is as high as 95.5%, and the SLC10A1 mutation spectrum is an absolute advantage. Neonatal hyperbilirubinemia, early infant cholestasis and pregnancy cholestasis patients with significant and persistent hyperbilirubinemia all require analysis of the SLC10A1 gene to rule out NTCP deficiency.
The existing SLC10A1 genotyping detection method comprises an enzyme digestion technology, a first-generation sequencing technology, a suspension magnetic bead liquid chip technology and the like. The enzyme digestion technology is used for carrying out enzyme digestion treatment on the corresponding PCR amplified fragments by using restriction enzymes, then gel electrophoresis is used for running gel, and the obtained bands are manually interpreted to distinguish different SLC10A1 genotypes. The method needs to purchase special enzyme, has high cost, complex detection reaction steps, long time consumption and easy error of manual interpretation; the first generation sequencing technology is long in time consumption and is not suitable for clinical large-scale popularization and use; the suspension magnetic bead liquid phase chip has higher technical cost, complex operation and longer detection time, and can influence the timeliness of clinical decisions.
Disclosure of Invention
Based on this, the object of the present invention is to provide a genotyping detection primer for SLC10A1 allele, which can simply, rapidly and efficiently distinguish 3 genotypes of SLC10A1 gene c.800C > T locus.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
In a first aspect of the invention, a SLC10A1 gene c.800C > T locus typing detection primer is provided, wherein the typing primer comprises an upstream primer and a downstream primer, the upstream primer is shown as SEQ ID NO.1, and the downstream primer is shown as SEQ ID NO. 2.
In a second aspect of the invention, a method for typing and detecting a locus c.800C > T of an SLC10A1 gene is provided, which comprises the following steps:
(1) Extracting DNA of a sample to be detected;
(2) Performing PCR amplification on the DNA using the typing detection primer as described above;
(3) HRM analysis was performed on the PCR amplified products.
In some of these embodiments, the PCR amplified system comprises the following components: a DNA template, the upstream primer, the downstream primer, dntps, a DNA polymerase, BSA, and a fluorescent dye.
In some of these embodiments, the fluorescent dye is EvaGreen.
In some of these embodiments, the final concentration of the upstream and/or downstream primers in the PCR amplification system is 0.05. Mu.M to 1. Mu.M, preferably 0.1. Mu.M to 0.3. Mu.M, more preferably 0.2. Mu.M.
In some of these embodiments, the PCR amplified system comprises the following concentrations of components: 10ng/μl-50ng/μl of DNA, 0.05 μM-1 μM of the upstream primer, 0.05 μM-1 μ M, dNTP 10 nM-20 nM of the downstream primer, 0.03U/μl-0.05U/μl of DNA polymerase, and 4ug/μl-6 ug/μl of BSA.
In some of these embodiments, the PCR amplification procedure is: 1) 3-5 min at 95 ℃; 2) 95 ℃ for 10s and 58 ℃ to 60 ℃ for 25s; repeating step 2) for 35-40 cycles.
In a third aspect of the invention, the application of the typing detection primer in preparing a detection kit for SLC10A1 gene c.800C > T site mutation related diseases is provided.
In some of these embodiments, the SLC10A1 gene c.800C > T site mutation associated disorder is sodium taurocholate cotransporter polypeptide deficiency.
In a fourth aspect of the invention, a kit for detecting a disease associated with mutation of a c.800C > T locus of an SLC10A1 gene is provided, and comprises the typing detection primer.
In some of these embodiments, the kit further comprises a DNA polymerase, dntps, fluorescent dye.
In some of these embodiments, the SLC10A1 gene c.800C > T site mutation associated disorder is sodium taurocholate cotransporter polypeptide deficiency.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides a parting detection primer for SLC10A1 gene c.800C & gtT locus, which has good amplifications for TT type (mutant homozygote), CC type (wild homozygote) and CT type (heterozygote) of SLC10A1 gene c.800C & gtT locus under the same amplification condition, can effectively improve the resolution of a melting curve in HRM detection, effectively distinguish TT type, CC type and CT type, and can simply, quickly and efficiently realize parting detection for SLC10A1 gene c.800C & gtT locus, and has high accuracy, good specificity, good repeatability and low cost.
Drawings
FIG. 1 is a representation of HRM melting peak for the genotyping of the SLC10A1 gene c.800C > T locus using primer 4 of the present invention.
Detailed Description
The experimental procedure of the present invention, in which no specific conditions are noted in the following examples, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
The following description is made with reference to specific embodiments.
Example 1
For the genotyping detection of 3 genotypes (TT type, CC type, CT type) of the SLC10A1 gene c.800C > T (p.Ser267Phe) locus, a large number of studies and screens were performed on a plurality of amplified regions, and a typing detection primer (primer 4 described below) capable of effectively distinguishing TT type, CC type, CT type was obtained.
In this example, the following 4 kinds of primers are taken as examples to compare and demonstrate the typing effect of the different primers.
Primer 1:
SLC-100-F(SEQ ID NO.1)：5’-ccaaaatgtccaactctgtt-3’；
SLC-100-R(SEQ ID NO.4)：5’-ctggaaaatcatgtagaggagg-3’；
Amplification product sequence 1(SEQ ID NO.5)：ccaaaatgtccaactctgttccaccatcctcaatgtggcctttccacctgaagtcattggaccacttttcttctttcccctcctctacatgattttccag.
Primer 2:
SLC-130-F(SEQ ID NO.6)：5’-cagcatggagactggatg-3’；
SLC-130-R(SEQ ID NO.7)：5’-cccttctccaagctggaa-3’；
amplification product sequence 2(SEQ ID NO.8)：cagcatggagactggatgccaaaatgtccaactctgttccaccatcctcaatgtggcctttccacctgaagtcattggaccacttttcttctttcccctcctctacatgattttccagcttggagaaggg.
Primer 3:
SLC-74-F(SEQ ID NO.9)：5’-aaaggccacattgagga-3’；
SLC-74-R(SEQ ID NO.10)：5’-aaagaagaaaagtggtcc-3’；
Amplicon sequence 3 (SEQ ID No. 11): AAAATGTCCAACTCTGTTCCACCATCCTCAATGTGGCCTTTCCACCTGAAGTCATTGGACCACTTTTCTTCTTT.
Primer 4:
SLC-44-F(SEQ ID NO.1)：5’-ccaaaatgtccaactctgtt-3’；
SLC-44-R(SEQ ID NO.2)：5’-gaaaggccacattgaggat-3’；
amplification product sequence 4 (SEQ ID NO. 3): CCAAAATGTCCAACTCTGTTCCACCATCCTCAATGTGGCCTTTC.
All primer pairs were synthesized by Shanghai Biotechnology services Inc.
The 4 primers are used for detecting samples with known genotypes, and the samples comprise 21 CC type samples, 20 CT type samples and 13 TT type samples.
The detection method comprises the following steps:
(1) Extracting sample DNA: the peripheral blood sample of the person to be tested is collected, and the DNA of the sample is extracted by using the blood genome DNA extraction kit which is a reagent, and the operation is strictly carried out according to the specification.
(2) The DNA extracted by the 4 primer pairs was amplified by PCR.
The total volume of the PCR amplification reaction system is 10 μl, and the system consists of the following components: genomic DNA (50 ng/. Mu.l) 1. Mu.l, 0.2. Mu.M upstream primer, 0.2. Mu.M downstream primer, dNTP 15nM, klen Taq0.4U, hemo KlenTaq Reaction Buffer. Mu.l, BSA (5 mg/ml) 1. Mu.l,(20×in water)1μl、RNase-Free ddH2O 3.65μ1。
The PCR amplification procedure was as follows: 1) 3 minutes at 95 ℃; 2) Step 2) was carried out for a total of 40 cycles at 95℃for 10 seconds, 60℃for 25 seconds.
(3) HRM analysis of PCR amplification products: HRM detection is carried out on PCR amplified products by using a Tianlong technology full-automatic medical PCR (Gentier R) instrument, and the method is specifically as follows: the process of melting the products of HRM analysis of PCR amplified products is: maintaining at 95deg.C for 1min, cooling to 40deg.C for 1min, heating from 40deg.C to 60deg.C, melting at 0.02 ℃/sec, heating to 90deg.C, maintaining for 1 sec, and cooling to 40deg.C for 30 sec; the signal is detected in real time in the process of heating and melting from 60 ℃ to 90 ℃, and the signal detection frequency is 10 times/DEG C.
Data were collected and the Tm values were read to determine genotype.
The genotype melting curve peak Tm values for the 4 primers resulted in the following:
primer 1: the Tm value range for CT type is: 78.92-79.11 ℃; tm value range for CC: 79.01-79.51 ℃; tm value range of TT type is: 78.67-79.00 ℃. The Tm values of CT type, CC type and TT type overlap, and CT type, CC type and TT type cannot be distinguished.
Primer 2: the Tm value range for CT type is: 80.34-80.45 ℃; tm value range for CC: 80.44-80.85 ℃; tm value range of TT type is: 80.06-80.24 ℃. The Tm values of CT type and CC type overlap, and CT type and CC type cannot be distinguished.
Primer 3: the Tm value range for CT type is: 78.26-78.39 ℃; tm value range for CC: 78.20-78.63 ℃; tm value range of TT type is: 78.10-78.31 ℃. The Tm values of CT type, CC type and TT type overlap, and CT type, CC type and TT type cannot be distinguished.
Primer 4: the Tm value range for CT type is: 72.64-73.13 ℃; tm value range for CC: 73.23-73.81 ℃; tm value range of TT type is: 72.53-72.62 ℃. The Tm values between CT type, CC type and TT type 3 do not have an overlap region, and can completely distinguish 3 genotypes.
Therefore, the primer 4 can effectively distinguish CT type, CC type and TT type of SLC10A1 gene c.800C > T locus, and the HRM melting peak value detected by the primer 4 is represented in figure 1.
Example 2
In order to verify the accuracy of the parting detection of the primer 4, 28 samples are collected, and the parting detection of the locus c.800C & gtT of the SLC10A1 gene is carried out by utilizing the primer 4; all samples were simultaneously subjected to SLC10A1 gene c.800C > T site sequencing analysis, samples were PCR amplified (the method was the same as in example 1), and the products were sent to Shanghai Bioengineering Co., ltd. For Sanger sequencing to verify the accuracy and specificity of the detection results.
The method for typing detection using the primer 4 is the same as in example 1, and specifically comprises the following steps:
(1) Extracting sample DNA: collecting peripheral blood samples of a person to be tested, extracting sample DNA by using a reagent blood genome DNA extraction kit, and carrying out operation strictly according to the specification.
(2) The extracted DNA was amplified by PCR using the primer 4.
The total volume of the PCR amplification reaction system is 10 μl, and the system consists of the following components: genomic DNA (50 ng/. Mu.l) 1. Mu.l, 0.2. Mu.M upstream primer, 0.2. Mu.M downstream primer, dNTP 15nM, klen Taq0.4U, hemo KlenTaq Reaction Buffer. Mu.l, BSA (5 mg/ml) 1. Mu.l,(20×in water)1μl、RNase-Free ddH2O 3.65μ1。
The PCR amplification procedure was as follows: 1) 3 minutes at 95 ℃; 2) Step 2) was carried out for a total of 40 cycles at 95℃for 10 seconds, 60℃for 25 seconds.
(3) HRM analysis of PCR amplification products: HRM detection of PCR amplified products is carried out by using a Tianlong technology full-automatic medical PCR (Gentier 96R) instrument, and the method is concretely as follows: the process of melting the products of HRM analysis of PCR amplified products is: maintaining at 95deg.C for 1min, cooling to 40deg.C for 1min, heating from 40deg.C to 60deg.C, melting at 0.02 ℃/sec, heating to 90deg.C, maintaining for 1 sec, and cooling to 40deg.C for 30 sec; and detecting signals in real time in the process of heating and melting from 60 ℃ to 90 ℃, wherein the signal detection frequency is 10 times/DEG C, and collecting data.
The Tm values of the respective samples were compared with Tm ranges of 3 genotypes of the primer 4 in example 1, and genotypes of the respective samples were obtained, the Tm ranges being as follows: the Tm value range for CT type is: 72.64-73.13 ℃; tm value range for CC: 73.23-73.81 ℃; tm value range of TT type is: 72.53-72.62 ℃.
The detection results are filled in Table 1 together with the sequencing results.
TABLE 1
The results show that the primer 4 can effectively distinguish CT type, CC type and TT type of SLC10A1 gene c.800C > T locus, the detection result is 100% identical with the sequencing result, and the accuracy is high.
The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
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| CN108823321A (en) * | 2018-06-25 | 2018-11-16 | 北京向中生物技术有限公司 | A kind of the HRM detection method and primer of beta-casein gene parting |
| WO2021156828A2 (en) * | 2020-02-06 | 2021-08-12 | Janssen Sciences Ireland Unlimited Company | Biomarkers and uses thereof in the treatment of chronic hepatitis b infection |
| CN116970685A (en) * | 2023-03-11 | 2023-10-31 | 广东省妇幼保健院（广东省妇产医院、广东省儿童医院） | qPCR kit for simultaneously detecting two mutation sites on one target gene and method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108823321A (en) * | 2018-06-25 | 2018-11-16 | 北京向中生物技术有限公司 | A kind of the HRM detection method and primer of beta-casein gene parting |
| WO2021156828A2 (en) * | 2020-02-06 | 2021-08-12 | Janssen Sciences Ireland Unlimited Company | Biomarkers and uses thereof in the treatment of chronic hepatitis b infection |
| CN116970685A (en) * | 2023-03-11 | 2023-10-31 | 广东省妇幼保健院（广东省妇产医院、广东省儿童医院） | qPCR kit for simultaneously detecting two mutation sites on one target gene and method thereof |
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| 李华 等: "一例钠牛磺胆酸共转运多肽缺陷病患儿临床和遗传学分析", 中国当代儿科杂志, no. 04, 2 April 2018 (2018-04-02) * |
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