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HPA allelic gene typing detection reagent kit

A technology for detecting kits and alleles, applied in the biological field, can solve the problems of complicated operation, non-specificity of specific primers, and inability to obtain results automatically, and achieve the effects of low experimental cost, improved objectivity and accuracy

Inactive Publication Date: 2012-06-06
上海血液生物医药有限责任公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Experimental defects: the results cannot be obtained automatically; a pair of alleles requires double-tube operation; the congenital deficiency of specific primers leads to non-specificity, and the accuracy needs to be improved
[0015] Experimental defects: hybridization temperature must be strictly controlled; washing conditions are strict; false positives and false negatives are prone to occur
[0019] Experimental defect: high experimental cost
[0023] Experimental defects: complex operation; enzymatic hydrolysis growth experiment time; some HPA alleles have no suitable enzyme cutting point
Since HPA is a biallelic co-dominant gene, Taq technology can be used for SNPs typing. At present, there is no relevant report on HPA system typing using this technology

Method used

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  • HPA allelic gene typing detection reagent kit
  • HPA allelic gene typing detection reagent kit
  • HPA allelic gene typing detection reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, preparation and assembly of each component of kit:

[0054] 1) Synthesis of primers and probes: The primers and probes were synthesized by ABI, an American Applied Biosystems company. The sequences of primers 1-12 are SEQ ID NO: 1-12, and the sequences of MGB-Taqman probes 1-12 are SEQ ID NO: 1-12. ID NO: 13-24, the specific sequence is shown in Table 2. Wherein the 3' end of the MGB probe is marked with a fluorescent quenching group TAMRA; and wherein the 5' end of the MGB-Taqman probes 1, 3, 5, 7, 9 and 11 is provided with a fluorescent reporter group FAM; wherein MGB- The 5' ends of Taqman probes 2, 4, 6, 8, 10 and 12 are equipped with a fluorescent reporter group VIC, and the mixture of primers and probes is diluted to: the concentration of the primer is 900nM, and the concentration of the probe is 200nM .

[0055] The sequence of the probe and primer used in the present embodiment of table 2

[0056]

[0057]

[0058] 2) Positive control and ...

Embodiment 2

[0061] Embodiment 2, the extraction of template DNA:

[0062] Eight samples used in this embodiment: sample 1 (abbreviated as Z1), sample 2 (abbreviated as Z2), sample 3 (abbreviated as Z3), sample 4 (abbreviated as Z4), sample 5 (abbreviated as Z5), sample Sample 6 (Z6 for short), sample 7 (Z7 for short), and sample 8 (Z8 for short) were derived from peripheral blood DNA of healthy blood donors in Shanghai, China, all of which were unrelated donors. Template DNA was prepared by salting out method.

[0063] After the template DNA was extracted, the template DNA was diluted to a concentration of 10 ng / μl with TE buffer.

Embodiment 3

[0064] Embodiment 3, real-time fluorescent quantitative PCR detection:

[0065] 1) Refer to Table 3 for the corresponding sample pattern control in each well of the 96-well reaction plate of the kit used in this example.

[0066] Table 3 Reaction plate pattern control list

[0067]

sample 1

sample 2

sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

Negative

control

Positive homozygous

Control 1

Positive homozygous

Control 2

Positive heterozygous

control

HPA-1

sample 1,

HPA-1

sample 2,

HPA-1

sample 3,

HPA-1

sample 4,

HPA-1

sample 5,

HPA-1

sample 6,

HPA-1

sample 7,

HPA-1

sample 8,

HPA-1

Negative pair

According to,

HPA-1

masculine pure and pair

According to 1,

HPA1

masculine and p...

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Abstract

The invention belongs to the technical field of biology, in particular to an HPA allelic gene typing detection reagent kit. The reagent kit of the invention comprises a primer and an MGB-probe. The reagent kit can judge the gene type of the allelic gene according to the observation of the difference of two kinds of fluorescence curves and the distribution of scattergrams after the amplification, so the HPA allelic gene typing detection can be completed. The reagent kit of the invention can meet the experiment requirement of high flux, and the whole typing experiment can be completed in two hours under the condition of the existence of proper DNA detection materials. The problem of fast systemic typing on HPA-1 to 5 and 15 on people groups in China can be solved, at the same time, the condition of homozygotes and heterozygotes can be identified, the typing results can be directly obtained, and the invention has the characteristics of flexibility, stability, accuracy and high efficiency.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an HPA allele typing detection kit. Background technique [0002] Platelets are non-nucleated blood cells. The main function of platelets is to promote hemostasis and accelerate coagulation. At the same time, platelets also have the function of maintaining the integrity of capillary walls. In the process of hemostasis and coagulation, platelets have the functions of forming thrombus, blocking wounds, and releasing various factors related to coagulation. Platelets play a central role in the formation of blood clots in the fast-flowing arterial and arteriolar system. [0003] Platelet membranes contain a variety of proteins, which are linked with a large number of carbohydrate branched chains to become glycoproteins. Platelet glycoprotein is not only very important to maintain the shape and integrity of platelets, but also constitutes various receptors of platelets, enabl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 冯明亮沈彤刘达庄赵玉林刘熔增
Owner 上海血液生物医药有限责任公司
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