Multiplex polymerase chain reaction (PCR) method and kit for detecting human RhD blood type genotypes

A genotype and kit technology, which is applied in the field of multiplex PCR and kits for detecting human RhD blood type genotypes, can solve the problems of inability to judge negative results, difficulty in interpretation, and cumbersome operations.

Inactive Publication Date: 2012-11-21
陕西省血液中心
View PDF1 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 1. Expensive: Although the D gene detection reagents produced by Germany’s BAG and INNO-TRAIN companies are reliable, they cost about 1200-1300 RMB per person, which is very expensive; A copy costs about 100 RMB, but the reliability is poor, and this price is still unaffordable for large-scale testing;
[0008] 2. The operation is cumbersome: the D gene detection reagents of Germany’s BAG and IVIN companies need 4 to 8 reaction tubes per person, while the American G&T reagents need 12 reaction tubes per person, and the consumption of sample DNA and consumables is very large , and the addition of samples is cumbersome, error-prone, and difficult to interpret, so it is not suitable for large-scale testing;
[0009] 3. The internal control is the human growth hormone gene, but in our actual use, we found that there are amplification bands in the internal control, but there is no amplification product in the detection band. It is impossible to judge whether some negative results are caused by the degradation of the Rh gene.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Multiplex polymerase chain reaction (PCR) method and kit for detecting human RhD blood type genotypes
  • Multiplex polymerase chain reaction (PCR) method and kit for detecting human RhD blood type genotypes
  • Multiplex polymerase chain reaction (PCR) method and kit for detecting human RhD blood type genotypes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] The design of embodiment 1 primer sequence

[0088] According to the primer sequence designed according to the specific site, the primer mixture comprises SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7 and SEQ ID NO8. Among them, SEQ ID NO1 and SEQ ID NO2 are the upstream and downstream primers for specifically amplifying the DEL gene located in the 9th exon, respectively, and the last base of SEQ ID NO1 is designed for the mutation of G>A at position 1227, and SEQ ID NO2 Located in the intron between the 9th and 10th exon, the length of the primer amplified fragment is 102bp; SEQ ID NO3 and SEQ ID NO4 are the upstream and downstream primers for amplifying the 8th exon of the RhD and RhCE genes respectively, Used as an internal control, the length of the amplified fragment is 206bp; SEQ ID NO5 and SEQ ID NO6 are the upstream and downstream primers for specifically amplifying the RhD gene located in the 10th exon, respectively, and the...

Embodiment 2

[0091] The PCR reaction of embodiment 2 single pairs of primers

[0092] The rationality of the primer design was verified by the PCR reaction of a single pair of primers, so as to finally determine the primer components that can be used in multiple PCR reactions.

[0093] The test materials were d / d, D / d and DEL type DNA, the reaction buffer was ordinary PCR buffer, Taq enzyme was purchased from Shanghai Promega Company, and dNTP was purchased from Beijing Dingguo Biotechnology Company. The reaction system is 20 μl: add 1.0 μl primer, 2.0 μl PCR buffer, 2.0 μl 25mM MgCl in a 0.5mL Ependoff tube 2 , 1.0 μl ldNTP mixture, 0.2 μl Taq DNA polymerase, 3.0 μl template DNA and 11.8 μl double distilled water, mix well;

[0094] The annealing temperature was determined according to the Tm value of the primers, and the reaction conditions were as follows:

[0095] SEQ ID NO1 and SEQ ID NO2: Denaturation at 94°C for 5 minutes, followed by 30 cycles of PCR reactions (94°C, 30 seconds, ...

Embodiment 3

[0101] Embodiment 3 The present invention is compared with other kits

[0102] In another embodiment of the present invention, the present invention is a kit for detecting human RhD blood genotype, which kit includes: 1 tube of primer mixture; 1 tube of PCR primer buffer; 1 tube of Taq polymerase; 1 tube of dNTP mixture tube; positive control 2 tubes, negative control 1 tube; and instructions for use; wherein, the PCR primer buffer contains 2.0 μl 500mM KCl, 2.0 μl 100mM Tris-HCl (PH 9.0), 2.6 μl 25mM MgCl 2 , 0.2μl 1.0M (NH 4 ) 2 SO 4 , 1.0 μl DMSO and 5.0 μl DDW. The DNA to be tested includes INNO-TRAIN’s standard quality control controls, D / d type and d / d type; according to the instruction manual, add 12.8 μl PCR primer buffer, 2.0 μl primer mix, 2.0 μl dNTP The mixture, 0.2 μl Taq DNA polymerase and 3.0 μl template DNA were mixed thoroughly; the reaction conditions were as follows: denaturation at 94°C for 5 minutes, and then 35 cycles of PCR reaction (94°C, 40 seconds...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a multiplex polymerase chain reaction (PCR) method capable of detecting human RhD blood type genotypes and an in vitro diagnostic kit prepared by applying the method and capable of detecting the human RhD blood type genotypes comprising D/D, D/d, DEL, DEL/d and d/d genotype. According to the method, variation of specific sequence loci of the different genotypes of a human RhD blood type is utilized to design a plurality of pairs of primers to perform a PCR. Accordingly, the multiplex PCR method and the in vitro diagnostic kit can be regarded to be on the basis of a PCR-SSP (sequence specific primer) principle. By designing the sequence specific primers according to different loci and by optimizing mixing proportion, reaction buffer solution ingredients and PCR reaction conditions of the sequence specific primers, the aim of accurately detecting the human RhD blood type genotypes is achieved. In an embodiment, the multiplex PCR method for detecting the human RhD blood type genotypes is provided, and in another embodiment, the kit for detecting the human RhD blood type genotypes is provided. Details of one or a plurality of embodiments are described in attached drawings and explanations. By reading the attached drawings, detailed descriptions and claims, other characteristics, aims and advantages of the multiplex PCR method and the kit can be learned about clearly.

Description

technical field [0001] The invention relates to a multiplex PCR method capable of detecting human RhD blood type genotypes, and an in vitro diagnostic kit prepared by applying the method, which can detect human RhD blood type genotypes, including D / D, D / d, DEL, DEL / d and d / d genotypes. technical background [0002] In this paper, the term "Rh" is the first two letters of the foreign language name of the rhesus monkey (Rhesus Macacus). Since the RhD antigen was discovered in 1939, it has become an important antigen in red blood cells after ABO blood type because it causes hemolytic disease of the newborn and extravascular delayed hemolytic transfusion reaction. The antigenicity of the Rh blood group is very strong, especially the D antigen is the strongest, second only to the A and B antigens of the ABO system. Those who have RhD antigen on human red blood cells are Rh positive, otherwise they are negative. The RhD-negative blood type accounts for about 3 to 5‰ of Chinese...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 徐华叶世辉吴大洲王满妮左琴琴段勇
Owner 陕西省血液中心
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products