Establishment of loop-mediated isothermal amplification method for detection of Legionella pneumophila / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a simple and rapid molecular detection for Legionella pneumophila.</p><p><b>METHODS</b>The loop-mediated isothermal amplification (LAMP) was applied for detection of Legionella pneumophila. A set of primers were designed to identify six special areas in mip gene of Legionella pneumophila. Genomic DNAs from 13 bacterial strains,including 8 Legionella pneumophila strains and 5 other bacterial strains were amplified by LAMP and general PCR method to evaluate the specificity and sensibility of LAMP.</p><p><b>RESULT</b>All positive tubes produced visible white precipitation, and no precipitation was observed in others. By adding smart green fluorescent dye, all Legionella pneumophila positive tubes presented a strong green fluorescence, while others showed weak fluorescence. The detection rate of LAMP was higher than that of general PCR. The detection limits were 576fg with genomic DNA of Legionella pneumophila,and 8 cfu/mL with positive water samples.</p><p><b>CONCLUSION</b>LAMP detection of Legionella pneumophila is an effective and low-cost method with high specificity and sensitivity requiring no special equipment.</p>

Sequence analysis of a novel HLA-A * 2459 allele / 中国实验血液学杂志

This study was aimed to investigate the molecular genetics basis of a novel allele HLA-A * 2459 in Chinese population. DNA was extracted from whole blood by PEL-FREEZ DNA extraction kit. The amplification of HLA-A exons 2 - 4 of the proband was preformed by allele specific primer PCR and the amplified product was sequenced bidirectionally with primers. The sequencing results showed HLA-A alleles of the proband as A * 1101 and the novel allele. The sequences of the novel allele have been submitted to GenBank (DQ313255, DQ313256, DQ313257). After Blast HLA analysis, the novel allele showed only one nucleotide differences with HLA-A * 24020101 at nucleotide position 527 T to C in exon 3. This results in an amino acid changes from Val to Ala at codon 152. In conclusion, this allele is a novel one and has been officially named HLA-A * 2459 by the WHO Nomenclature Committee.

Analysis of natural killer cell immunoglobulin-like receptor gene family in HLA-identical sibling / 中国实验血液学杂志

The aim of study was to analyze natural killer immunoglobulin (Ig)-like receptor (KIR) gene content in HLA-identical sibling and to investigate the possibility of their KIR match. Samples were genotyped for HLA by Luminex method and polymerase chain reaction sequence based typing, the KIR gene was detected by polymerase chain reaction sequence-specific primers. The results showed that 17 KIR genes could be observed in the 27 pairs HLA-A, -B, -Cw and -DRB1 locus identical sibling samples. All individuals contained KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2.; 20 different KIR genotypes and 12 haplotypes have been found, the most common KIR genotypes was 2,2 with frequency 29.6% and KIR haplotype was 2 with frequency 53.0%. The A KIR haplotype was the most prevalent with frequency 67.2%; 12 pairs (44.4%) HLA identical sibling donor-recipients showed KIR match in genotype and haplotype, 13 pairs (48.1%) with one KIR haplotype mismatch and 2 pairs (7.4%) with two KIR haplotype mismatch; 1 pair was matched between donor KIR2DL1 and patient HLA-Cw (Lys80) ligand, 17 pairs were matched between KIR2DL2/KIR2DL3 and HLA-Cw (Asn80) ligand, 5 pairs were matched between KIR3DL1 and HLA-Bw4 ligand. It is suggested that the probability of KIR mismatch is high in HLA-identical sibling.

Identification and sequence analysis of a null HLA-B allele HLA-B*5408N newly detected / 中国实验血液学杂志

The study was purposed to investigate the molecular genetic basis for HLA novel allele HLA-B*5408N in Chinese population. DNA was extracted from whole blood by commercial DNA extraction kit, the HLA-B exons 2 - 4 of the proband was amplified by allele specific primers PCR and the amplified product was sequenced for exons 2, 3 and 4 bidirectionally. The sequencing results showed HLA-B alleles of the proband as B*1527 and the novel allele. The sequences of the novel allele have been submitted to Genbank (DQ295998, DQ295999, DQ296000). After blast analysis, the novel allele showed a single nucleotide mismatch with HLA-B*5401 in exon 3 at position 553 G-->T, which resulted in an amino acid changing from Glu to premature stop codon at position 161. No the HLA-B54 antigen specificity expression in the proband cells was found using HLA-AB serological Typing Trays. It is concluded that this allele is a novel null allele and has been officially named B*5408N by the WHO Nomenclature Committee.

Sequence analysis of a novel HLA-DRB1 allele, DRB1 * 1212 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the molecular genetics basis for HLA novel allele HLA-DRB1*1212 in Chinese population.</p><p><b>METHODS</b>Genomic DNA was extracted from whole blood by salting-out method. HLA-DRB1 gene exon 2 was amplified by PCR with group-specific primers from genomic DNA. PCR products were cut back from agarose gels and purified to sequence directly. The polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSO) was performed to confirm the mutations which were detected by sequencing in this study.</p><p><b>RESULTS</b>The sequencing results showed HLA-DRB1 alleles of the proband as DRB1*090102 and the novel allele. The sequences of the novel allele have been submitted to GenBank (AY899825). Through BLAST analysis, the novel allele was found to be different from DRB1*120101 at position 199A-->C in exon 2, that results in an amino acid change from Ile to Leu at codon 67.</p><p><b>CONCLUSION</b>This allele is a novel and has been officially named as DRB1*1212 by the WHO Nomenclature Committee.</p>

Allelic polymorphism analysis of killer cell immunoglobulin-like receptor gene 3DL2 in Zhejiang Han population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the allelic polymorphism of killer cell immunoglobulin (Ig)-like receptor (KIR) gene 3DL2 in Zhejiang Han population.</p><p><b>METHODS</b>Allelic definition within KIR3DL2 was achieved through using the PCR-sequence specific oligonucleotide probes (PCR-SSOP) method and the specific amplification of exon 3-exon 4 and exon 8-exon 9. Twenty-one digoxigenin-labelled probes were used to the SSOP analysis.</p><p><b>RESULTS</b>Seven alleles of KIR3DL2 were observed, in which KIR3DL2 *002 had the highest allele frequency, 0.57. One sample could not be given the allele combination according to the probe profile.</p><p><b>CONCLUSION</b>The PCR-SSOP method was reliable. There is a distinctive allele frequency of KIR3DL2 in Zhejiang Han population.</p>

Establishment of delta block matching technique / 中国实验血液学杂志

To establish delta block HLA-matching technique, DNA was extracted from whole blood by salting-out method, delta block was amplified by polymerase chain reaction (PCR), and PCR product was detected by GeneScan. The results showed that delta block had polymorphism in 104 samples without sibship of the Han people from Zhejiang province. The range of DNA fragment length was 81-393 bp and could be divided into 4 groups: 81-118 bp, 140-175 bp, 217-301 bp, 340-393 bp. The numbers of DNA fragments were 6-32. It is concluded that the method of delta block matching is reliable and can be applied to select donors for the patients to be transplanted. It is the first time to get delta block data of the Han people in China.

Sequence analysis of HLA-B*4061 allele newly found / 中国实验血液学杂志

The aim of this study was aimed to investigate the molecular genetic basis for a novel HLA allele, HLA-B*4061, in Chinese population. DNA was extracted from whole blood by salting-out method. The HLA-B exons 1 - 8 of the proband was amplified and the amplified product was cloned using TOPO TA cloning sequencing kit to split the two alleles apart. Both strands of exons 2, 3 and 4 of chosen colonies were sequencing. The PCR-SSP was performed to confirm the mutations detected by sequencing. The sequencing results showed HLA-B alleles of the proband as B*4601 and the novel allele. The sequences of the novel allele have been submitted to GenBank (DQ089628, DQ089629, DQ089630). After HLA blast analysis, the novel allele showed a single nucleotide mismatch with B*400101 in exon 2 at position 272 C-->A, as the results, changing amino acid from Ser to Tyr at codon 67. It is concluded that this allele is a novel one and has been officially named B*4061 by the WHO Nomenclature Committee.

Identification and sequence analysis of a novel HLA-B*5614 allele / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the molecular genetics basis for a novel HLA allele, HLA-B*5614, in Chinese population.</p><p><b>METHODS</b>DNA was extracted from whole blood by salting-out method. The HLA-B exons 2-4 of the proband was amplified and the amplified product was cloned using TOPO cloning sequencing kit to split the two alleles apart. Both strands of exons 2,3 and 4 of chosen colonies were sequencing. The PCR-SSP was performed to confirm the mutations detected by sequencing.</p><p><b>RESULTS</b>The sequencing results showed the HLA-B alleles of the proband as B*1502 and the novel allele. The sequences of the novel allele have been submitted to GenBank (AY601726, AY610727, AY610728). After BLAST analysis, the novel allele differs from B*5608 by a single nucleotide at position 277G-->C in exon 2. This results in an amino acid change from Gly to Arg at codon 93.</p><p><b>CONCLUSION</b>This allele is a novel allele and has been officially named B*5614 by the WHO Nomenclature Committee.</p>

Establishment of beta block matching technique / 中国实验血液学杂志

The purpose of this study was to establish beta block matching technique. DNA was extracted from whole blood by salting-out method, beta block matching was performed by PCR and GeneScan technique. The results showed that the length of fragments amplificated in 100 samples was different and the range of them was 91-197 bp. Amplification fragments could be divided into four regions: 91-93, 105-113, 125-139 and 177-197 bp respectively. 91 bp DNA fragments could be found in all of samples. The numbers of DNA fragments with different length have been shown high polymorphism and they focused on the range of seven to twenty four. In conclusion, the beta block matching technique is reliable and applicable to the selection of hematopoietic stem cell transplantation donors.

Polymorphism of killer cell immunoglobulin-like receptors gene family in Zhejiang Han population / 中国实验血液学杂志

To analyze killer immunoglobulin (Ig)-like receptor (KIR) gene content and allelic polymorphism in Zhejiang Han population, samples were genotyped by polymerase chain reaction sequence-specific primers (PCR-SSP). The results demonstrated that all 17 KIR genes could be observed in the population. All individuals contained 2DL4, 3DL2 and 3DL3 genes. The frequencies of these genes was 1.00. 2DL1, 2DL3, 2DP1, 3DP1*003, 3DL1, 2DS4*001/002 loci were more common, their frequencies were 0.902, 0.902, 0.902, 0.902, 0.7598, 0.5615 respectively, while the frequencies of 2DL2, 2DL5A, 2DL5B, 2DS1, 2DS2, 2DS3, 2DS4*003, 2DS5, 3DS1 and 3DP1*001/002 were relatively lower. The A KIR haplotype was the most prevalent (74.7%) in Zhejiang Han population and there were 12 different KIR haplotypes in total, the most common was 2 (53.0%). Twenty six different genotypes have been found in the population, AJ (2, 2) and AF (1, 2) showed higher frequencies, followed by AH (2, 5), NN2 (2, 6), AI (1, 5) and AG (1, 1). Fifteen of these genotypes have not been found in Caucasians so far and four new KIR profiles could not be assigned to the haplotypes according to standard assign method. In conclusion, there are distinctive frequencies of KIR gene content, haplotype as well as genotype in Zhejiang Han population.