[HLA genotyping by automatic semi-quantitative polymerase chain reaction-reverse sequence specific oligonucleotide].
OBJECTIVE
To explore a HLA genotyping method that can be used for organ transplantation tissue typing, and especially for establishing hemopoietic stem cell bank and the cord blood stem cell bank by analyzing the PCR-DNA.


METHODS
Modified automatic method based on semi-quantitative amplification system of HLA-I, I allele genotyping was established using reverse sequence-specific oligonucleotide with polymerase chain reaction (PCR-RSSO), and was compared with PCR (using sequence specific primer, PCR-SSR) and manual PCR-RSSO in terms of the accuracy, resolution, and the quantity of DNA consumption. A total of 635 blood samples were genotyped with HLA-A, B, C, DR and DQ alleles using auto semi-quantitative PCR-RSSO, 166 of which were also examined using PCR-SSP and manual PCR-RSSO simultaneously. RDSULTS: The success rate of automatic semi-quantitative PCR-RSSO, PCR-SSP and manual PCR-RSSO was 98.4% (3 124/3 175), 98.8% (656/664) and 88.3% (732/830) respectively, with no significant difference between the former 2 as indicated by X2 test (P>0.05). Auto semi-quantitative PCR-RSSO, however, yielded significantly higher success rate than manual PCR-RSSO (P<0.05).


CONCLUSIONS
PCR-RSSO is capable of identifying 706 alleles of HLA-I, II antigens which amounts to 75.43% of the 936 alleles published by WHO in 2000, with intermediate to high resolution, even in the case of the homozygote. The hybridization results documenting the original data can be conveniently preserved. This method therefore possesses the merits of low expenses, low labor intensity, and rapid processing of large number of samples.
Xiao
Lu-lu
LL
Guangzhou Tissue Typing Centre, Guangzhou 510095, China.
Hao
Gui-qin
GQ
Ye
Xin
X
Chen
Hong-tao
HT
Tan
Yin
Y
Zhang
Sheng
S
D000483:Alleles; D004247:DNA / Q000302:isolation & purification; D005838:Genotype; D015395:Histocompatibility Antigens Class I / Q000145:classification / Q000235:genetics*; D000949:Histocompatibility Antigens Class II / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D004247:DNA