US 12,630,876 B2Granted

Kit for genotyping of platelet and neutrophil antigens and glycoproteins

AssigneeShanghai Blood Center

InventorsLuyi Ye, Huijun Zhu, Ruishu Li, Min Fu, Ping Lu, Ziyan Zhu

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Abstract

The present invention provides a mass spectrometry-based method and a kit for genotyping of platelet and neutrophil antigens and glycoproteins, which are used for genotyping of platelet-specific antigens, platelet CD36 glycoproteins and neutrophil antigens; by designing an optimal primer combination, problems such as homologous sequences and rich GC are overcome, moreover, by improving amplification reaction conditions and using nucleic acid mass spectrometry as a platform, 35 platelet-specific antigen polymorphic sites, 10 CD36 polymorphic sites and 8 neutrophil antigen polymorphic sites can be simultaneously detected in 2 reactions. The present invention has the characteristics of high specificity and sensitivity, and fast and high throughput, and can be used in clinic, scientific research, platelet donor routine screening, etc.

Claims7

Dependent claims

A method for genotyping by mass spectrometry detection comprises the following steps:

(1) using an amplification primers mix to amplify genes from a DNA template by a multiplex PCR reaction;
(2) purifying an amplification product obtained in Step (1) by an alkaline phosphatase;
(3) using an extension primers mix to extend and amplify the purified product in Step (2) by a single base;
and
(4) conducting sample application on a single-base extended product obtained in Step (3) onto a chip for mass spectrometry detection;
wherein the amplification primers mix and the extension primers are included in a tube, and the amplification primers and the extension primers are as below:
Forward
Reverse
Extension
SNP sites
primers
primers
primers
CD36 (1)
rs550565800
SEQ ID NO:
1
SEQ ID NO:
2
SEQ ID NO:
3
CD36 (2)
rs75326924
SEQ ID NO:
4
SEQ ID NO:
5
SEQ ID NO:
6
CD36 (3)
rs572295823
SEQ ID
SEQ ID
SEQ ID
NO:
7
NO:
8
NO:
9
CD36 (4)
rs201355711
SEQ ID
SEQ ID
SEQ ID
NO:
10
NO:
11
NO:
12
CD36 (5)
rs148910227
SEQ ID
SEQ ID
SEQ ID
NO:
13
NO:
14
NO:
15
CD36 (6)
rs201765331
SEQ ID
SEQ ID
SEQ ID
NO:
16
NO:
17
NO:
18
CD36 (7)
rs545489204
SEQ ID
SEQ ID
SEQ ID
NO:
19
NO:
20
NO:
21
CD36 (8)
rs142186404
SEQ ID
SEQ ID
SEQ ID
NO:
22
NO:
23
NO:
24
CD36 (9)
rs201759307
SEQ ID
SEQ ID
SEQ ID
NO:
25
NO:
26
NO:
27
CD36 (10)
rs767892046
SEQ ID
SEQ ID
SEQ ID
NO:
28
NO:
29
NO:
30.

114 more elements

Dependencies are inferred from the claim text.

Facts

Application: 18/156,739Filed 19 Jan 2023
Priority: CN 202210098023.526 Jan 2022
Published: US 2023/0235390 A127 Jul 2023
Granted: 19 May 2026
Term ends: Not estimatedThe source records don’t support an estimate.

Classification10 codes

C12Q 1/6872
Methods for sequencing: involving mass spectrometry
C12Q 1/6806
Involving nucleic acids: preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6853
Nucleic acid amplification reactions: using modified primers or templates
C12Q 1/6858
Nucleic acid amplification reactions: allele-specific amplification