Hyperbilirubinemia gene fluorescence PCR melting curve method detection kit and method

Abstract

The invention discloses a hyperbilirubinemia gene fluorescent PCR (polymerase chain reaction) melting curve method detection kit. Comprising a nucleic acid reaction solution, a DNA polymerase mixed solution, a W1 primer probe reagent, a W2 primer probe reagent, a W3 primer probe reagent, a W4 primer probe reagent, a W5 primer probe reagent, a wild type quality control product A, a wild type quality control product B, a wild type quality control product C, a wild type quality control product D, a wild type quality control product E and a negative quality control product, according to the kit, different mutation types of 12 sites of multiple genes of a sample can be detected by directly carrying out melting curve analysis once after PCR amplification is finished, the whole operation is completed within 3 hours, the consumed time is short, and the kit is very suitable for clinical timeliness and rapidness requirements.

Description

Technical Field

[0001] The invention relates to the technical field of hyperbilirubinemia diagnosis, in particular to a hyperbilirubinemia gene fluorescence PCR melting curve method detection kit and method. Background Art

[0002] Neonatal hyperbilirubinemia is one of the common diseases of newborns. It affects most newborns due to elevated unconjugated bilirubin and is the main reason for hospitalization in the first week after birth.

[0003] There are many types of gene mutations in patients with hyperbilirubinemia. In recent years, research on the treatment and medication of neonatal hyperbilirubinemia has developed rapidly. Taking the UGT1A1 gene c.211G>A mutation (Gilbert syndrome, GS) as an example, GS is an autosomal recessive genetic disease with a prevalence of about 3% to 13% in the global population. When GS children have hemolytic diseases or hypothyroidism in the neonatal period, they are at higher risk of severe hyperbilirubinemia, kernicterus, and even death. The occurrence and development of neonatal jaundice after discharge from the hospital largely depends on the visual observation of parents. Therefore, jaundice often continues to develop due to inaccurate visual observation, and even causes severe neonatal hyperbilirubinemia. High levels of bilirubin that last for about 5 days can cause irreversible effects on the development of the neonatal nervous system, and untimely treatment can also lead to other more serious consequences.

[0004] This patent screens the following genetic diseases that cause hyperbilirubinemia: (1) Congenital non-hemolytic jaundice (Gilbert syndrome): Due to the insufficient activity of bilirubin glucuronyl transferase in the microsomal organelles of hepatocytes, which is hereditary or acquired, the normal conjugation reaction of unconjugated bilirubin in hepatocytes is affected, so that the uptake of bilirubin by hepatocytes is also hindered, resulting in a double defect in the uptake and conjugation function of hepatocytes for unconjugated bilirubin. From a strict definition, it is characterized by non-hemolytic, unconjugated hyperbilirubinemia, while serum bile acid and liver function are normal. The typical UGT1A1 gene mutation in Gilbert syndrome patients not only affects the metabolic process of bilirubin, but also participates in the metabolic pathway of related drugs, which may lead to a decrease or even disappearance of enzyme activity, hindering the normal metabolic process of bilirubin, bilirubin accumulation and causing neonatal hyperbilirubinemia. Common mutation sites are: c.211G>A: Mutations at this site can cause the 71st amino acid of the UGT1A1 protein to change from glycine to arginine, reducing the activity of the UGT1A1 enzyme, thereby affecting the metabolic process of drugs and substances such as bilirubin, and leading to changes in the concentration of related substances in the body; c.686C>A: Changes at this site will cause changes in the encoded amino acid, affecting the structure and function of the UGT1A1 enzyme, and ultimately affecting its metabolic activity, which is related to the susceptibility to certain diseases and individual differences in drug metabolism; c.1091C>T: Mutations at this site can affect the metabolic process of drugs such as acetaminophen, leading to the accumulation of drug metabolites in the body and increasing the risk of adverse reactions. Special attention should be paid when using related drugs; c.1456T>G: It will cause changes in the protein structure encoded by the UGT1A1 gene, affecting the function of the UGT1A1 enzyme, and then affecting its ability to metabolize substrates. It may play a certain role in drug metabolism and the occurrence and development of certain diseases. c.-41_-40dup: If the child is found to carry a mutation at the c.-41_-40dup site, it will help to clarify whether the cause of jaundice is related to abnormal bilirubin metabolism caused by reduced UGT1A1 enzyme activity. (2) Glucose-6-phosphate dehydrogenase (G6PD) deficiency. Common mutation sites are: c.1376G>T: This is one of the more common mutation sites in southern China. This mutation causes changes in the structure and function of the G6PD protein, resulting in reduced enzyme activity, which in turn causes G6PD deficiency; c.1388G>A.This mutation also affects the normal function of G6PD protein, resulting in decreased enzyme activity; c.95A>G: can cause changes in the amino acid sequence of G6PD protein, thereby affecting the stability and activity of the enzyme and increasing the patient's sensitivity to oxidative stress; c.871G>A: can cause changes in the amino acid sequence of G6PD protein, thereby affecting the stability and activity of the enzyme; c.1024C>T: causes changes in the amino acids of the translated G6PD protein, resulting in changes in its spatial conformation and stability, affecting the active center or key domain, and reducing enzyme activity. (3) Intrahepatic cholestasis / Citrin deficiency: Citrin deficiency is a type of autosomal recessive inherited disease that includes two major diseases: adult-onset type II citrullinemia and neonatal intrahepatic cholestasis caused by citrin deficiency. The causative gene SLC25A13 of this disease is located on chromosome 7q21.3, and the protein encoded is called citrin protein. Abnormalities in the SLC25A13 gene can lead to a deficiency in the encoded citrin protein and the occurrence of adult-onset type II citrullinemia or neonatal intrahepatic cholestasis caused by citrin protein deficiency. Common mutation sites are: c.851del4: This is one of the most common mutation sites in the SLC25A13 gene, also known as IVS16ins3kb. The mutation at this site refers to the insertion of a fragment of about 3kb into the 16th intron of the gene, resulting in abnormalities in the transcription and translation of the gene, thereby affecting the normal synthesis and function of the citrin protein. (4) Sodium taurocholate cotransporting polypeptide (NTCP) deficiency: NTCP deficiency is a new hereditary bile acid metabolism disease that has gradually been recognized in recent years and is an autosomal recessive genetic disease. NTCP deficiency is caused by mutations in the SLC10A1 gene, which affects the function of NTCP to absorb bile salts from plasma, resulting in a large amount of bile acid accumulation in the blood, forming a significant and stubborn hypercholecidemia. Common mutation sites are: Among the reported NTCP deficiency patients, SLC10A1 gene mutation, c.800C>T is the most common. Adult patients with this disease generally lack positive symptoms and signs, but some patients are diagnosed with pregnancy cholestasis and even choose cesarean section. Some NTCP deficiency children show neonatal hyperbilirubinemia or early infant cholestasis. Currently, the CFDA-approved kits for genetic testing of hyperbilirubinemia have relatively single detection items and cannot detect multiple genes at the same time. Secondly, there are mainly the following technologies for genetic testing of hyperbilirubinemia. (1) PCR-sequencing method: high cost, long time, cumbersome operation, and high technical requirements (2) Fluorescence PCR method: Fluorescence PCR is the most widely used gene detection platform. It is simple to operate and closed-tube reaction, which can effectively avoid contamination of amplification products, but there is only one probe in a single reaction tube, and the detection throughput is small. (3) Gene chip method: complex operation, long process, high cost of reagents and instruments, easy to cause contamination results, and there is a certain possibility of false positives.(4) Nucleic acid mass spectrometry: The equipment and reagents are expensive, the result analysis is complex, and the requirements for equipment and personnel are high. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the above technical defects, provide a hyperbilirubinemia gene fluorescence PCR melting curve method detection kit and method, and solve the above problems.

[0006] In order to solve the above problems, the technical scheme of the present invention is a hyperbilirubinemia gene fluorescent PCR melting curve method detection kit: including nucleic acid reaction solution, DNA polymerase mixture, W1 primer probe reagent, W2 primer probe reagent, W3 primer probe reagent, W4 primer probe reagent, W5 primer probe reagent, wild-type quality control product A, wild-type quality control product B, wild-type quality control product C, wild-type quality control product D, wild-type quality control product E, negative quality control product;

[0007] The W1 primer probe reagent includes: upstream and downstream primers and probes corresponding to the G6PD-rs137852327 site, upstream and downstream primers and probes corresponding to the UGT1A1-rs4148323 site, and upstream and downstream primers and probes corresponding to the SLC10A1-rs2296651 site; the W2 primer probe reagent includes: upstream and downstream primers and probes corresponding to the UGT1A1-rs35350960 site, upstream and downstream primers and probes corresponding to the G6PD-rs137852342 site, and upstream and downstream primers and probes corresponding to the UGT1A1-rs34946978 site. Primers and probes; the W3 primer probe reagent includes: upstream and downstream primers and probes corresponding to the GT1A1-rs34993780 site, upstream and downstream primers and probes corresponding to the G6PD-rs72554665 site, and upstream and downstream primers and probes corresponding to the G6PD-rs137852340 site; the W4 primer probe reagent includes: upstream and downstream primers and probes corresponding to the G6PD-rs72554664 site and the SLC25A13-c.851del4 site; the W5 primer probe reagent includes upstream and downstream primers and probes corresponding to the UGT1A1c.-41_-40dup rs3064744 site.

[0008] Further, the upstream primer sequence corresponding to the G6PD-rs137852327 site of the W1 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the G6PD-rs137852327 site is 5'-3', the probe sequence corresponding to the G6PD-rs137852327 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the G6PD-rs137852327 site is VIC; the upstream primer sequence corresponding to the UGT1A1-rs4148323 site of the W1 primer probe reagent is 5'-3', and the downstream primer sequence corresponding to the UGT1A1-rs4148323 site is 5'-3', the probe sequence corresponding to the UGT1A1-rs4148323 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the UGT1A1-rs4148323 site is ROX; the upstream primer sequence corresponding to the SLC10A1-rs2296651 site of the W1 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the SLC10A1-rs2296651 site is 5'-3', the probe sequence corresponding to the SLC10A1-rs2296651 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the SLC10A1-rs2296651 site is CY5.

[0009] Further, the upstream primer sequence corresponding to the UGT1A1-rs35350960 site of the W2 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the UGT1A1-rs35350960 site is 5'-3', the probe sequence corresponding to the UGT1A1-rs35350960 site is 5'-3', the fluorescent group labeled with the probe corresponding to the UGT1A1-rs35350960 site is FAM, the upstream primer sequence corresponding to the G6PD-rs137852342 site of the W2 primer probe reagent is 5'-3', and the downstream primer sequence corresponding to the G6PD-rs137852342 site is The sequence of the probe corresponding to the G6PD-rs137852342 site is 5'-3', the fluorescent group labeled with the probe corresponding to the G6PD-rs137852342 site is ROX, the upstream primer sequence corresponding to the UGT1A1-rs34946978 site of the W2 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the UGT1A1-rs34946978 site is 5'-3', the probe sequence corresponding to the UGT1A1-rs349469780 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the UGT1A1-rs34946978 site is CY5.

[0010] Further, the W3 primer probe reagent UGT1A1-rs34993780 site corresponding to the upstream primer sequence is 5'-3', the UGT1A1-rs34993780 site corresponding to the downstream primer sequence is 5'-3', the UGT1A1-rs34993780 site corresponding to the probe sequence is 5'-3', the UGT1A1-rs34993780 site corresponding to the probe labeled fluorescent group is VIC, the W3 primer probe reagent G6PD-rs72554665 site corresponding to the upstream primer sequence is 5'-3', the G6PD-rs72554665 site corresponding to the downstream primer sequence is 5'-3'. The W3 primer probe reagent has an upstream primer sequence of 5'-3', a downstream primer sequence of 5'-3', a probe sequence corresponding to the G6PD-rs72554665 site is 5'-3', and a fluorescent group labeled with the probe corresponding to the G6PD-rs72554665 site is ROX. The W3 primer probe reagent has an upstream primer sequence of 5'-3', a downstream primer sequence of 5'-3', a probe sequence corresponding to the G6PD-rs137852340 site is 5'-3', and a fluorescent group labeled with the probe corresponding to the G6PD-rs137852340 site is CY5.

[0011] Further, the upstream primer sequence corresponding to the G6PD-rs72554664 site of the W4 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the G6PD-rs72554664 site is 5'-3', the probe sequence corresponding to the G6PD-rs72554664 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the G6PD-rs72554664 site is VIC, and the upstream primer sequence corresponding to the SLC25A13-c.851del4 site of the W4 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the SLC25A13-c.851del4 site is 5'-3', the probe sequence corresponding to the SLC25A13-c.851del4 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the SLC25A13-c.851del4 site is ROX.

[0012] Furthermore, the upstream primer sequence corresponding to the UGT1A1c.-41_-40dup rs3064744 site of the W5 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the UGT1A1c.-41_-40dup rs3064744 site is 5'-3', the probe sequence corresponding to the UGT1A1c.-41_-40dup rs3064744 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the UGT1A1c.-41_-40duprs3064744 site is FAM.

[0013] Furthermore, the wild-type quality control product A is a wild-type amplified target gene linear plasmid containing G6PD-rs137852327 site, UGT1A1-rs4148323 site, and SLC10A1-rs2296651 site; the wild-type quality control product B is a wild-type amplified target gene linear plasmid containing UGT1A1-rs35350960 site, G6PD-rs137852342 site, and UGT1A1-rs34946978 site. ; Wild-type quality control product C is a wild-type amplified target gene linear plasmid containing UGT1A1-rs34993780 site, G6PD-rs72554665 site, and G6PD-rs137852340 site; Wild-type quality control product D is a wild-type amplified target gene linear plasmid containing G6PD-rs72554664 site and SLC25A13-c.851del4 site; Wild-type quality control product E is a wild-type amplified target gene linear plasmid containing UGT1A1c.-41_-40dup rs3064744 site; Negative quality control products are tris(hydroxymethylaminomethane)-hydrochloric acid and ethylenediaminetetraacetic acid.

[0014] Further, the method of the hyperbilirubinemia gene fluorescent PCR melting curve detection kit is characterized by comprising the following steps: the upstream and downstream primers and probes are designed using Premier Biosoft's BeaconDesigner8 software, and the TaqManDesign in the BeaconDesigner8 software is used for design, "NewSequence" imports the site sequence to be designed, "AddSNPs" adds SNP site information, "TaqManProbeSearch" designs the primer probe of the site to be tested, and "ExportTaqManResults" exports the designed primer probe sequence information; the upstream and downstream primers and probes use the Primer-BLAST function of NCBI to perform specific analysis on the primer probe sequence designed for the site to be tested. Open the Primer-BLAST page of NCBI, fill in the primer sequence according to the prompts of the function interface operation, select the genome information, select the species information, click "GetPrimers", and obtain the comparison result. The comparison result shows that the homology with other sequences is low and the specificity is good. Each primer probe is synthesized by solid phase phosphoramidite triester chemical method, the primer is purified by PAGE after synthesis, and the probe is purified by HPLC after synthesis;

[0015] The preparation method of the wild-type quality control product A, wild-type quality control product B, wild-type quality control product C, wild-type quality control product D, and wild-type quality control product E is as follows: synthesize plasmids by using a full gene synthesis method, transfer the synthesized plasmids to Escherichia coli for cloning culture, extract the cultured Escherichia coli for plasmids, and obtain the desired plasmids; measure the plasmid concentration of the obtained plasmids with a Nanodropone ultra-micro spectrophotometer, calculate the corresponding copy number, and then dilute with 1×TE to the desired copy number; the calculation formula for the copy number of the plasmid stock solution is: copy number (copies / μL) = 6.02×10 14 × plasmid concentration (ng / μL) / (plasmid length×660); the working concentration of the plasmid used for the wild-type quality control product A, wild-type quality control product B, wild-type quality control product C, wild-type quality control product D, and wild-type quality control product E is E7.

[0016] Furthermore, the method of the hyperbilirubinemia gene fluorescent PCR melting curve detection kit comprises the following steps: 95°C 15s→58°C 45s, 50 cycles, wherein fluorescence is collected at 58°C 45s; 95°C 2min→40°C 5min→40°C~80°C, wherein the heating rate at 40°C~80°C is 0.04°C / s, melting curve analysis is performed and fluorescence is collected.

[0017] The advantages of the present invention compared with the prior art are:

[0018] 1. The detection throughput of the present invention is high: multiple fluorescently labeled probes can be designed simultaneously in a single reaction tube, and each probe can detect target gene sequences with different matching degrees, thereby greatly improving the detection throughput; PCR amplification and melting curve analysis can be performed on the same instrument, reducing the operation steps, thereby reducing manpower investment and saving experimental consumables. 3. The detection of the present invention is short in time: a melting curve analysis can be directly performed once after the PCR amplification to detect different mutation types of 12 sites of multiple genes in the sample. The entire operation is completed within 3 hours, which is short in time and very suitable for clinical timeliness and quickness requirements. 4. The closed tube reaction of the present invention is not easy to be contaminated: PCR amplification and melting curve analysis are completed in one step, without the need for post-PCR operation steps, and the entire detection process is carried out in a closed reaction tube to avoid amplification products from contaminating the experimental environment. 5. The present invention has strong specificity: the fluorescent probe can achieve highly specific binding with the target sequence, greatly reducing non-specific signals. And the detection results are interpreted in the form of the melting peak (i.e., melting point) of the melting curve, which is clear and easy to understand, not prone to errors, and can achieve high specificity of detection.

[0019] Instruction Manual

[0020] Figure 1 It is the result of the jaundice project of the present invention Figure 1(UGT1A1c.-41_-40dup rs3064744 wild type).

[0021] Figure 2 It is the result of the jaundice project of the present invention Figure 2 (UGT1A1c.-41_-40dup rs3064744 homozygous).

[0022] Figure 3 It is the result of the jaundice project of the present invention Figure 3 (UGT1A1c.-41_-40dup rs3064744 heterozygous type).

[0023] Figure 4 It is the result of the jaundice project of the present invention Figure 4 (G6PD-c.871G>A wild type).

[0024] Figure 5 It is the result of the jaundice project of the present invention Figure 5 (G6PD-c.871G>A homozygous).

[0025] Figure 6 It is the result of the jaundice project of the present invention Figure 6 (G6PD-c.871G>A heterozygous).

[0026] Figure 7 It is the result of the jaundice project of the present invention Figure 7 (UGT1A1-c.211G>A wild type).

[0027] Figure 8 It is the result of the jaundice project of the present invention Figure 8 (UGT1A1-c.211G>A homozygous).

[0028] Fig. 9 It is the result of the jaundice project of the present invention Figure 9 (UGT1A1-c.211G>A heterozygous).

[0029] Fig.10 It is the result of the jaundice project of the present invention Figure 10 (SLC10A1-c.800C>T wild type).

[0030] Fig.11 It is the result of the jaundice project of the present invention Figure 10 1 (SLC10A1-c.800C>T homozygous).

[0031] Fig.12 It is the result of the jaundice project of the present invention Figure 10 2 (SLC10A1-c.800C>T heterozygous).

[0032] Fig.13 It is the result of the jaundice project of the present invention Figure 10 3 (UGT1A1-c.686C>T wild type).

[0033] Fig.14 It is the result of the jaundice project of the present invention Figure 10 2 (UGT1A1-c.686C>T homozygous).

[0034] Fig.15 It is the result of the jaundice project of the present invention Figure 10 5. (UGT1A1-c.686C>T heterozygous).

[0035] Fig.16 It is the result of the jaundice project of the present invention Figure 10 VI (G6PD-c.1024C>T wild type).

[0036] Fig.17 It is the result of the jaundice project of the present invention Figure 10 Seven (G6PD-c.1024C>T homozygous).

[0037] Fig.18 It is the result of the jaundice project of the present invention Figure 10 8 (G6PD-c.1024C>T heterozygous).

[0038] Fig.19 It is the result of the jaundice project of the present invention Figure 10 IX (UGT1A1-c.1091C>T wild type).

[0039] Fig. 20 It is the result of the jaundice project of the present invention Figure 2 10 (UGT1A1-c.1091C>T homozygous).

[0040] Fig.21 It is the result of the jaundice project of the present invention Figure 2 11 (UGT1A1-c.1091C>T heterozygous).

[0041] Fig. 22 It is the result of the jaundice project of the present invention Figure 2 12 (UGT1A1-C.1456T>G wild type).

[0042] Fig.23 It is the result of the jaundice project of the present invention Figure 2 Thirteen (UGT1A1-C.1456T>G homozygous).

[0043] Fig.24 It is the result of the jaundice project of the present invention Figure 2 Twelve (UGT1A1-C.1456T>G heterozygous).

[0044] Fig.25 It is the result of the jaundice project of the present invention Figure 2 15 (G6PD-c.1376G>T wild type).

[0045] Fig.26 It is the result of the jaundice project of the present invention Figure 2 Sixteen (G6PD-c.1376G>T homozygous).

[0046] Fig. 27 It is the result of the jaundice project of the present invention Figure 2 Seventeen (G6PD-c.1376G>T heterozygous).

[0047] Fig.28 It is the result of the jaundice project of the present invention Figure 2 XVIII (G6PD-c.95A>G wild type).

[0048] Fig.29 It is the result of the jaundice project of the present invention Figure 2 Nineteen (G6PD-c.95A>G homozygous).

[0049] Fig.30 It is the result of the jaundice project of the present invention Figure 3 10 (G6PD-c.95A>G heterozygous).

[0050] Fig.31 It is the result of the jaundice project of the present invention Figure 3 XI (G6PD-c.1388G>A wild type).

[0051] Fig.32 It is the result of the jaundice project of the present invention Figure 3 Twelve (G6PD-c.1388G>A homozygous).

[0052] Fig.33 It is the result of the jaundice project of the present invention Figure 3 Thirteen (G6PD-c.1388G>A heterozygous).

[0053] Fig.34 It is the result of the jaundice project of the present invention Figure 3 Twelve (SLC25A13-c.851del4 wild type).

[0054] Fig.35 It is the result of the jaundice project of the present invention Figure 3 Fifteen (SLC25A13-c.851del4 homozygous).

[0055] Fig.36 It is the result of the jaundice project of the present invention Figure 3 Sixteen (SLC25A13-c.851del4 heterozygous).

[0056] Fig.37 It is the result of the jaundice project of the present invention Figure 3 Seventeen (UGT1A1-c.-41Δ-40dup wild type).

[0057] Fig.38 It is the result of the jaundice project of the present invention Figure 3 Eighteen (UGT1A1-c.-41_-40dup homozygous).

[0058] Fig.39 It is the result of the jaundice project of the present invention Figure 3 Nineteen (UGT1A1-c.-41_-40dup heterozygous). DETAILED DESCRIPTION

[0059] In order to make the contents of the present invention more clearly understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0060] like Figure 1-39 As shown in the figure, the fluorescent PCR melting curve method detection kit for hyperbilirubinemia gene includes nucleic acid reaction solution, DNA polymerase mixture, W1 primer probe reagent, W2 primer probe reagent, W3 primer probe reagent, W4 primer probe reagent, W5 primer probe reagent, wild-type quality control product A, wild-type quality control product B, wild-type quality control product C, wild-type quality control product D, wild-type quality control product E, and negative quality control product; W1 primer probe reagent includes: upstream and downstream primers and probes corresponding to G6PD-rs137852327 site, upstream and downstream primers and probes corresponding to UGT1A1-rs4148323 site, and upstream and downstream primers and probes corresponding to SLC10A1-rs2296651 site; W2 primer probe reagent includes: upstream and downstream primers corresponding to UGT1A1-rs35350960 site The W3 primer probe reagents include: upstream and downstream primers and probes corresponding to the GT1A1-rs34993780 site, upstream and downstream primers and probes corresponding to the G6PD-rs72554665 site, and upstream and downstream primers and probes corresponding to the G6PD-rs137852340 site; the W4 primer probe reagents include: upstream and downstream primers and probes corresponding to the G6PD-rs72554664 site and the SLC25A13-c.851del4 site; the W5 primer probe reagents include upstream and downstream primers and probes corresponding to the UGT1A1c.-41_-40duprs3064744 site. The DNA polymerase is a hot-start DNA polymerase, MightyAmp from Takara. TMGenotyping Kit。

[0061] The upstream primer sequence corresponding to the G6PD-rs137852327 site of the W1 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the G6PD-rs137852327 site is 5'-3', the probe sequence corresponding to the G6PD-rs137852327 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the G6PD-rs137852327 site is VIC; the upstream primer sequence corresponding to the UGT1A1-rs4148323 site of the W1 primer probe reagent is 5'-3', and the downstream primer sequence corresponding to the UGT1A1-rs4148323 site is 5'- 3', the probe sequence corresponding to the UGT1A1-rs4148323 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the UGT1A1-rs4148323 site is ROX; the upstream primer sequence corresponding to the SLC10A1-rs2296651 site of the W1 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the SLC10A1-rs2296651 site is 5'-3', the probe sequence corresponding to the SLC10A1-rs2296651 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the SLC10A1-rs2296651 site is CY5. The upstream primer sequence corresponding to the UGT1A1-rs35350960 site of the W2 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the UGT1A1-rs35350960 site is 5'-3', the probe sequence corresponding to the UGT1A1-rs35350960 site is 5'-3', the fluorescent group labeled with the probe corresponding to the UGT1A1-rs35350960 site is FAM, the upstream primer sequence corresponding to the G6PD-rs137852342 site of the W2 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the G6PD-rs137852342 site is 5 '-3', the probe sequence corresponding to the G6PD-rs137852342 site is 5'-3', the fluorescent group labeled with the probe corresponding to the G6PD-rs137852342 site is ROX, the upstream primer sequence corresponding to the UGT1A1-rs34946978 site of the W2 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the UGT1A1-rs34946978 site is 5'-3', the probe sequence corresponding to the UGT1A1-rs349469780 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the UGT1A1-rs34946978 site is CY5.The upstream primer sequence corresponding to the UGT1A1-rs34993780 site of the W3 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the UGT1A1-rs34993780 site is 5'-3', the probe sequence corresponding to the UGT1A1-rs34993780 site is 5'-3', the fluorescent group labeled by the probe corresponding to the UGT1A1-rs34993780 site is VIC, the upstream primer sequence corresponding to the G6PD-rs72554665 site of the W3 primer probe reagent is 5'-3', and the downstream primer sequence corresponding to the G6PD-rs72554665 site is The W3 primer probe reagent has an upstream primer sequence of 5'-3' for the G6PD-rs137852340 site and a downstream primer sequence of 5'-3' for the G6PD-rs137852340 site. The probe sequence for the G6PD-rs72554665 site is 5'-3', and the fluorescent group labeled with the probe for the G6PD-rs72554665 site is ROX. The W3 primer probe reagent has an upstream primer sequence of 5'-3' for the G6PD-rs137852340 site and a downstream primer sequence of 5'-3' for the G6PD-rs137852340 site. The probe sequence for the G6PD-rs137852340 site is 5'-3', and the fluorescent group labeled with the probe for the G6PD-rs137852340 site is CY5. The upstream primer sequence corresponding to the G6PD-rs72554664 site of the W4 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the G6PD-rs72554664 site is 5'-3', the probe sequence corresponding to the G6PD-rs72554664 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the G6PD-rs72554664 site is VIC. The upstream primer sequence corresponding to the SLC25A13-c.851del4 site of the W4 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the SLC25A13-c.851del4 site is 5'-3', the probe sequence corresponding to the SLC25A13-c.851del4 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the SLC25A13-c.851del4 site is ROX. The upstream primer sequence corresponding to the UGT1A1c.-41_-40dup rs3064744 site of the W5 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the UGT1A1c.-41_-40dup rs3064744 site is 5'-3', the probe sequence corresponding to the UGT1A1c.-41_-40dup rs3064744 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the UGT1A1c.-41_-40dup rs3064744 site is FAM.

[0062] The wild-type quality control product A is a wild-type amplified target gene linear plasmid containing G6PD-rs137852327, UGT1A1-rs4148323, and SLC10A1-rs2296651; the wild-type quality control product B is a wild-type amplified target gene linear plasmid containing UGT1A1-rs35350960, G6PD-rs137852342, and UGT1A1-rs34946978; the wild-type quality control product C is a wild-type amplified target gene linear plasmid containing UGT1A1-rs34993780 The wild-type control product D is the wild-type amplified target gene linear plasmid of the G6PD-rs72554664 site and the SLC25A13-c.851del4 site; the wild-type control product E is the wild-type amplified target gene linear plasmid of the UGT1A1c.-41_-40duprs3064744 site; the negative control products are tris(hydroxymethylaminomethane)-hydrochloric acid and ethylenediaminetetraacetic acid. The upstream and downstream primers and probes were designed using Premier Biosoft's BeaconDesigner8 software. The TaqManDesign in BeaconDesigner8 software was used for design. "New Sequence" was used to import the site sequence to be designed, "Add SNPs" was used to add SNP site information, "TaqMan Probe Search" was used to design the primer probe for the site to be tested, and "Export TaqMan Results" was used to export the designed primer probe sequence information. The upstream and downstream primers and probes used the Primer-BLAST function of NCBI to perform specificity analysis on the primer probe sequences designed for the site to be tested. Open the Primer-BLAST function page of NCBI, fill in the primer sequence according to the prompts of the function interface, select the genome information, select the species information, and click "Get Primers" to obtain the comparison results. The comparison results show that the homology with other sequences is low and the specificity is good. Each primer probe is synthesized by a solid phase phosphoramidite triester chemical method, the primers are purified by PAGE after synthesis, and the probes are purified by HPLC after synthesis;

[0063] The preparation methods of wild-type quality control products A, wild-type quality control products B, wild-type quality control products C, wild-type quality control products D, and wild-type quality control products E are as follows: synthesize plasmids using the whole gene synthesis method, transfer the synthesized plasmids to Escherichia coli for cloning culture, extract the cultured Escherichia coli for plasmids, and obtain the desired plasmids; measure the plasmid concentration of the obtained plasmids using a Nanodropone ultra-micro spectrophotometer, calculate the corresponding copy number, and then use 1×TE to dilute to the required copy number; the calculation formula for the copy number of the plasmid stock solution is: copy number (copies / μL) = 6.02×10 14 × plasmid concentration (ng / μL) / (plasmid length×660); the working concentration of the plasmid used for the wild-type quality control product A, wild-type quality control product B, wild-type quality control product C, wild-type quality control product D, and wild-type quality control product E is E7.

[0064] The reaction procedure of the kit includes the following steps: 95℃15s→58℃45s, 50 cycles, collecting fluorescence at 58℃45s; 95℃2min→40℃5min→40℃~80℃, where the heating rate of 40℃~80℃ is 0.04℃ / s, performing melting curve analysis and collecting fluorescence. The amplification primers for the sites are as follows:

[0065]

[0066]

[0067] Among them, F represents the upstream primer; R represents the downstream primer. The probe for the polymorphic site is as follows:

[0068]

[0069]

[0070] Sample DNA extraction: According to the actual situation of the sample, choose a suitable method to extract the sample DNA. The recommended method is the magnetic bead method. It should be noted that when used for fragment amplification, the DNA content in each reaction must reach 0.1-2.0ng. The amplification system is prepared, and the single-well system is prepared as shown in the following table:

[0071]

[0072] The machine reaction program settings are shown in the following table:

[0073]

[0074] Set the reaction volume to 20μL. Quality control: The wild-type control and negative control in the kit must meet the following conditions at the same time. Otherwise, the test results are invalid and need to be retested. The wild-type control has melting peaks in all twelve channels, and the Tm value of the melting peak in each channel is within the following range: as shown in the following table:

[0075]

[0076]

[0077] The negative control had no melting peak in each channel.

[0078] Result interpretation. Wild type: The sample to be tested has only wild-type melting peaks in twelve detection channels, and the Tm difference with the wild-type reference melting peak is within the range of ±1°C. Heterozygous: The sample to be tested has two melting peaks in a certain detection channel, one of which has a Tm value within the range of ±1°C with the wild-type reference melting peak, and the other has a Tm value above ±2°C with the wild-type reference melting peak. Homozygous: The sample to be tested has only one melting peak in a certain detection channel, and the Tm value of the melting peak of the wild-type reference is above ±2°C, and the Tm values ​​of the melting peaks of the remaining detection channels are within the range of ±1°C with the wild-type reference melting peak. Compound heterozygous: The sample to be tested has both mutant melting peaks in two or more detection channels, and the Tm value of the melting peak of the wild-type reference is above ±2°C; and wild-type melting peaks, and the Tm value of the melting peak of the wild-type reference is within the range of ±1°C. If the sample to be tested has no melting peak in any channel, it indicates that there is a problem in the sample extraction process, the test result is invalid, and it needs to be re-extracted and tested. If the re-test result has melting peaks in all test channels, the re-test result will be used as the final test result; if the re-test result is consistent with the initial test result and there is no melting peak in any test channel, the sample is invalid and needs to be re-collected.

Claims

1. A fluorescent PCR melting curve method detection kit for hyperbilirubinemia gene, characterized in that: Including nucleic acid reaction solution, DNA polymerase mixture, W1 primer probe reagent, W2 primer probe reagent, W3 primer probe reagent, W4 primer probe reagent, W5 primer probe reagent, wild-type quality control product A, wild-type quality control product B, wild-type quality control product C, wild-type quality control product D, wild-type quality control product E, negative quality control product; The W1 primer probe reagent includes: upstream and downstream primers and probes corresponding to the G6PD-rs137852327 site, upstream and downstream primers and probes corresponding to the UGT1A1-rs4148323 site, and upstream and downstream primers and probes corresponding to the SLC10A1-rs2296651 site; The W2 primer probe reagent includes: upstream and downstream primers and probes corresponding to the UGT1A1-rs35350960 site, upstream and downstream primers and probes corresponding to the G6PD-rs137852342 site, and upstream and downstream primers and probes corresponding to the UGT1A1-rs34946978 site; The W3 primer probe reagent includes: upstream and downstream primers and probes corresponding to the GT1A1-rs34993780 site, upstream and downstream primers and probes corresponding to the G6PD-rs72554665 site, and upstream and downstream primers and probes corresponding to the G6PD-rs137852340 site; The W4 primer probe reagent includes: upstream and downstream primers and probes corresponding to the G6PD-rs72554664 site and the SLC25A13-c.851del4 site; The W5 primer probe reagent includes upstream and downstream primers and probes corresponding to the UGT1A1 c.-41_-40dup rs3064744 site.

2. The hyperbilirubinemia gene fluorescent PCR melting curve detection kit and method according to claim 1, characterized in that: The DNA polymerase is a hot-start DNA polymerase, which is MightyAmp from Takara. TM GenotypingKit.

3. The hyperbilirubinemia gene fluorescent PCR melting curve method detection kit according to claim 1, characterized in that: The upstream primer sequence corresponding to the G6PD-rs137852327 site of the W1 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the G6PD-rs137852327 site is 5'-3', the probe sequence corresponding to the G6PD-rs137852327 site is 5'-3', and the fluorescent group labeled by the probe corresponding to the G6PD-rs137852327 site is VIC; the upstream primer sequence corresponding to the UGT1A1-rs4148323 site of the W1 primer probe reagent is 5'-3', and the downstream primer sequence corresponding to the UGT1A1-rs4148323 site is 5' -3', the probe sequence corresponding to the UGT1A1-rs4148323 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the UGT1A1-rs4148323 site is ROX; the upstream primer sequence corresponding to the SLC10A1-rs2296651 site of the W1 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the SLC10A1-rs2296651 site is 5'-3', the probe sequence corresponding to the SLC10A1-rs2296651 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the SLC10A1-rs2296651 site is CY5.

4. The hyperbilirubinemia gene fluorescent PCR melting curve method detection kit according to claim 1, characterized in that: The upstream primer sequence corresponding to the UGT1A1-rs35350960 site of the W2 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the UGT1A1-rs35350960 site is 5'-3', the probe sequence corresponding to the UGT1A1-rs35350960 site is 5'-3', the fluorescent group labeled with the probe corresponding to the UGT1A1-rs35350960 site is FAM, the upstream primer sequence corresponding to the G6PD-rs137852342 site of the W2 primer probe reagent is 5'-3', and the downstream primer sequence corresponding to the G6PD-rs137852342 site is 5'-3', the probe sequence corresponding to the G6PD-rs137852342 site is 5'-3', the fluorescent group labeled with the probe corresponding to the G6PD-rs137852342 site is ROX, the upstream primer sequence corresponding to the UGT1A1-rs34946978 site of the W2 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the UGT1A1-rs34946978 site is 5'-3', the probe sequence corresponding to the UGT1A1-rs349469780 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the UGT1A1-rs34946978 site is CY5.

5. The hyperbilirubinemia gene fluorescent PCR melting curve method detection kit according to claim 1, characterized in that: The W3 primer probe reagent has an upstream primer sequence of 5'-3' corresponding to the UGT1A1-rs34993780 site, a downstream primer sequence of 5'-3' corresponding to the UGT1A1-rs34993780 site, a probe sequence of 5'-3' corresponding to the UGT1A1-rs34993780 site, and a fluorescent group labeled with the probe corresponding to the UGT1A1-rs34993780 site is VIC. The W3 primer probe reagent has an upstream primer sequence of 5'-3' corresponding to the G6PD-rs72554665 site, and a downstream primer sequence of 5'-3' corresponding to the G6PD-rs72554665 site. The sequence of the probe corresponding to the G6PD-rs72554665 site is 5'-3', the fluorescent group labeled with the probe corresponding to the G6PD-rs72554665 site is ROX, the upstream primer sequence corresponding to the G6PD-rs137852340 site in the W3 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the G6PD-rs137852340 site is 5'-3', the probe sequence corresponding to the G6PD-rs137852340 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the G6PD-rs137852340 site is CY5.

6. The hyperbilirubinemia gene fluorescent PCR melting curve method detection kit according to claim 1, characterized in that: The upstream primer sequence corresponding to the G6PD-rs72554664 site of the W4 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the G6PD-rs72554664 site is 5'-3', the probe sequence corresponding to the G6PD-rs72554664 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the G6PD-rs72554664 site is VIC. The upstream primer sequence corresponding to the SLC25A13-c.851del4 site of the W4 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the SLC25A13-c.851del4 site is 5'-3', the probe sequence corresponding to the SLC25A13-c.851del4 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the SLC25A13-c.851del4 site is ROX.

7. The hyperbilirubinemia gene fluorescent PCR melting curve method detection kit according to claim 1, characterized in that: The upstream primer sequence corresponding to the UGT1A1 c.-41_-40dup rs3064744 site of the W5 primer probe reagent is 5'-3', the downstream primer sequence corresponding to the UGT1A1 c.-41_-40dup rs3064744 site is 5'-3', the probe sequence corresponding to the UGT1A1 c.-41_-40dup rs3064744 site is 5'-3', and the fluorescent group labeled with the probe corresponding to the UGT1A1 c.-41_-40dup rs3064744 site is FAM.

8. The hyperbilirubinemia gene fluorescent PCR melting curve method detection kit according to claim 1, characterized in that: The wild-type quality control product A is a wild-type amplified target gene line containing G6PD-rs137852327 site, UGT1A1-rs4148323 site, and SLC10A1-rs2296651 site; the wild-type quality control product B is a wild-type amplified target gene line containing UGT1A1-rs35350960 site, G6PD-rs137852342 site, and UGT1A1-rs34946978 site. Linear plasmid; wild-type quality control product C is a wild-type amplified target gene linear plasmid containing UGT1A1-rs34993780 site, G6PD-rs72554665 site, and G6PD-rs137852340 site; wild-type quality control product D is a wild-type amplified target gene linear plasmid containing G6PD-rs72554664 site and SLC25A13-c.851del4 site; wild-type quality control product E is a wild-type amplified target gene linear plasmid containing UGT1A1 c.-41_-40duprs3064744 site; negative quality control products are tris(hydroxymethylaminomethane)-hydrochloric acid and ethylenediaminetetraacetic acid.

9. The method of the hyperbilirubinemia gene fluorescent PCR melting curve method detection kit according to claims 1-8, characterized in that: The following steps are involved: The upstream and downstream primers and probes were designed using BeaconDesigner8 software from Premier Biosoft. The design was performed using TaqManDesign in BeaconDesigner8 software, "New Sequence" was used to import the site sequence to be designed, "Add SNPs" was used to add SNP site information, "TaqManProbeSearch" was used to design the primers and probes for the sites to be tested, and "Export TaqManResults" was used to export the designed primer and probe sequence information; The upstream and downstream primers and probes use the Primer-BLAST function of NCBI to perform specific analysis on the primer probe sequences designed for the test site. Open the Primer-BLAST function page of NCBI, fill in the primer sequence according to the prompts of the function interface, select the genome information, select the species information, and click "Get Primers" to get the comparison results. The comparison results show that the homology with other sequences is low and the specificity is good. Each primer probe is synthesized by solid phase phosphoramidite triester chemical method, the primer is purified by PAGE after synthesis, and the probe is purified by HPLC after synthesis; The preparation method of the wild-type quality control product A, wild-type quality control product B, wild-type quality control product C, wild-type quality control product D, and wild-type quality control product E is as follows: synthesize plasmids by using a full gene synthesis method, transfer the synthesized plasmids to Escherichia coli for cloning culture, extract the cultured Escherichia coli for plasmids, and obtain the desired plasmids; measure the plasmid concentration of the obtained plasmids with a Nanodropone ultra-micro spectrophotometer, calculate the corresponding copy number, and then dilute with 1×TE to the desired copy number; the calculation formula for the copy number of the plasmid stock solution is: copy number (copies / μL) = 6.02×10 14 × plasmid concentration (ng / μL) / (plasmid length×660); the working concentration of the plasmid used for the wild-type quality control product A, wild-type quality control product B, wild-type quality control product C, wild-type quality control product D, and wild-type quality control product E is E7.

10. The reaction procedure of the hyperbilirubinemia gene fluorescent PCR melting curve method detection kit according to claim 9, characterized in that: The method comprises the following steps: 95°C 15s→58°C 45s, 50 cycles, wherein fluorescence is collected at 58°C 45s; 95°C 2min→40°C 5min→40°C-80°C, wherein the heating rate at 40°C-80°C is 0.04°C / s, melting curve analysis is performed and fluorescence is collected.

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