Supplementary value of denaturing high performance liquid chromatography for routine prenatal diagnosis of spinal muscular atrophy by multiple ligation-dependent probe amplification / 中华医学遗传学杂志

Objective@#To explore the feasibility of high performance liquid chromatography (DHPLC) combined with multiple ligation-dependent probe amplification (MLPA) for the prenatal diagnosis of spinal muscular atrophy (SMA).@*Methods@#Three families who had given birth to children with SMA type I were subjected to prenatal diagnosis. Peripheral blood samples were collected from the three couples, and 10 mL amniotic fluid was taken for each fetus through amniocentesis at 16-24 gestational week. Following DNA extraction, maternal contamination was excluded by STR analysis. Copy numbers of the SMN genes were detected by denaturing high performance liquid chromatography (DHPLC). Relative copy number of SMN1, SMN2 and reference genes was detected with a MLPA P021 assay kit.@*Results@#The three couples were all found to harbor heterozygous deletion of exon 7 of the SMN1 gene by DHPLC. MLPA analysis also suggested that the three couples were all carriers of SMA mutations. The fetus of family 1 harbored homozygous deletion of exons 7 and 8 of the SMN1 gene, in addition with heterozygous deletion of exons 7 and 8 of the SMN2 gene, suggesting that the fetus had SMA. The fetus of family 2 also harbored homozygous deletion of exons 7 and 8 of the SMN1 gene, while the copy number of SMN2 gene was normal, suggesting that the fetus was a SMA patient too. The fetus of family 3 harbored heterozygous deletion of exons 7 and 8 of the SMN1 gene, in addition with heterozygous deletion of exons 7 and 8 of the SMN2 gene, suggesting that the fetus was a carrier.@*Conclusion@#DHPLC can effectively screen carriers of SMA mutations. Combined DHPLC and MLPA can provide accurate diagnosis for fetuses with a high risk for SMA.

Genetype distribution of mycoplasma pneumoniae by denaturing high-performance liquid chromatography / 中国小儿急救医学

Objective To investigate the genetype distribution of mycoplasma pneumoniae(MP) by denaturing high-performance liquid chromatography(DHPLC).Methods A total of 300 cases nasopharyngeal aspirate were collected from our hospital.The MP genes of standard strains and clinical specimens isolates were amplified by PCR followed by DHPLC and genetype determination.Results A total of 110 cases were positive after 24 hours fermentation from 300 cases with pharyngeal swab.By the specific primers of MP-129,MP-FH standard strain and specimens,2 280 bp and 2 580 bp gene fragments were made out respectively.One hundred and ten strains of clinical isolates were detected by DHPLC.One hundred and seven strains of P1-Ⅰ were 1b subtype,3 were type P1-Ⅱ which were all 2a subtype.Conclusion The genetype of MP infection in children from our hospital is P1-Ⅰ,1b subtype by using DHPLC technology.

Rapid diagnosis of trisomy 21 syndrome by denaturing high-performance liquid chromatography / 国际检验医学杂志

Objective To detect the double DNA of trisomy 21 syndrome patients by denaturing high-performance liquid chro-matography(DHPLC) in order to rapidly diagnose trisomy 21 syndrome .Methods To amplify DNA fragments of three short tan-dem repeats of D21S111 ,D21S1411 and D21S1412 using corresponding primers .Then DHPLC was introduced to analyze the DNA fragments in the temperature of 50 ℃ .Results DHPLC elution profiles of D21S111 ,D21S1411 and D21S1412 of normal control showed one peak or two peaks with the same altitude .However DHPLC elution profiles of 21 trisomy syndrome patients showed two peaks of different altitudes ,which one′s altitude was twice than another .Conclusion DHPLC is a sensitive ,convenient ,auto-matic and highly-efficient method to diagnose trisomy 21 syndrome and can be widely used in the clinic diagnosis .

Novel multiplex primer extension and denaturing high-performance liquid chromatography for genotyping of the deafness gene mutations / 中南大学学报(医学版)

To find a rapid and accurate genotyping method for specific non-syndromic hearing loss (NSHL)-causing gene mutations for disease diagnosis in different ethnic populations.Methods We performed a novel multiplex primer extension (PE) reaction in combination with denaturing high-performance liquid chromatography (DHPLC) to simultaneously detect and genotype the 6 most common mutations in 180 patients with NSHL (GJB2-235delC,GJB2-299delAT,PDS-A2168G,PDS IVS7-2A ＞ G,mtDNA-A1555G,and mtDNA-C1494T) in Chinese population.This method involved the amplification of the target sequence,followed by a purification step,a multiplex PE reaction,and DHPLC analysis performed on the Transgenomic Wave DNA fragment analysis system under fully-denaturing conditions.Results In a blind analysis,this technique successfully and accurately genotyped 100％ of the samples simultaneously characterized by direct sequencing.Conclusion Combination of PE and DHPLC is simple,rapid,accurate,and cost-effective for genotyping common disease-causing mutations,including substitutions,insertions,and deletions in NSHL,and may be successfully used in other genetic diseases.

Analysis of TP53 Gene Mutations in the Korean Patients with Lung Cancer

BACKGROUND: Lung cancer is one of the leading causes of cancer-related deaths throughout the world. The gene tumor protein 53 (TP53) is frequently mutated in cases of lung cancer. This study was performed to investigate the frequencies and types of mutations in the TP53 gene in Korean patients with lung cancer. METHODS: We obtained tissue samples from 80 lung cancer patients and synthesized TP53 cDNA by using RNA isolated from these tissues by performing reverse transcriptase polymerase chain reaction. Hybridization and denaturing high-performance liquid chromatography were performed to identify the TP53 gene mutations, and then, the mutations were validated by direct sequencing. RESULTS: Forty mutations out of the 80 patients (50.0%) were noted in the TP53 gene. The frequencies of TP53 gene mutation for different cancer types, namely, squamous cell carcinoma, adenocarcinoma, and small cell carcinoma were 61.1%, 27.3%, and 26.7%, respectively. The mutation frequencies in the different regions of the gene were 10.0% for exon 4, 35.0% for exon 5, 12.5% for exon 6, 22.5% for exon 7, 17.5% for exon 8, and 2.5% for exon 9. The frequently mutated positions were codon 179 in exon 5, codons 202 and 220 in exon 6, and codons 266 and 273 in exon 8. CONCLUSIONS: Exon 5 was the most frequently mutated region in the TP53 gene. Compared to the patients with the other types of cancers, patients with squamous cell carcinoma showed a higher frequency of TP53 mutation. Codon 179 was the most frequently mutated codon in the TP53 gene.

Establishment of the denaturing high-performance liquid chromatography combined with multiplex nucleic acid amplification method for rapid identification of four important pathogenic mycobacteria / 中国人兽共患病学报

A new molecular method for simultaneously rapid detection and differentiation of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium paratuberculosis was established by using denaturing high-performance liquid chromatography (DHPLC) combined with multiplex nucleic acid amplification. These 4 important pathogenic mycobacteria were identified by separation of 4 specific PCR-amplified target fragments by DHPLC analysis. A total of 51 Mycobacterium strains and 22 other bacterial species were tested to confirm the specificity of the multiplex PCR-DHPLC assay. The sensitivity of the assay was as low as 10~2-10~3 gene copies. This method rapidly identify the positive clinical samples from human and bovine with higher detection ratio than traditional culture method and was able to identify simultaneously four pathogenic Mycobacterium, which provided a new molecular tool for rapid detection of tuberculosis and paratuberculosis in human and animals.

PAX4 gene polymorphism and islet autoantibody-negative ketosis-prone diabetes / 中南大学学报(医学版)

Objective To investigate PAX4 gene polymorphism and its association with islet autoantibody-negative patients with ketosis-prone diabetes in Chinese Han population. Methods We screened the variation of exon 3 and 9 within PAX4 gene by denaturing high performance liquid chromatography(DHPLC) in 112 non-diabetes control subjects (NC group) and 141 patients with ketosis-prone diabetes (KPD group), who were both negative for glutamic acid decarboxylase antibody (GAD-Ab) as well as protein tyrosine phosphatase antibody (IA-2Ab) . The sequences of abnormal peaks were analyzed by DNA-sequencing. The A1168C single nucleotide polymorphism in PAX4 gene was genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 308 non-diabetic control subjects and 141 KPD patients. Results No variation was discovered in PAX4 gene exon 3 both in the patients and in the controls. There was a single nucleotide polymorphism locus A1168C in the PAX4 gene exon 9, which induced mis-sence mutation P321H(rs712701). No significant difference was observed in the genotype and allele frequencies of A1168C polymorphism between KPD patients and control subjects (P=0.532, 0.426). The difference was detected in the CC genotype and C allele frequencies in the KPD group when patients were stratified by gender (P=0.009,0.028). According to age at diagnosis, the difference was observed in the CC genotype and C allele frequencies between <20 years old and ≥20 years old in the KPD group (P=0.034,0.032). The level of FCP in the CC genotype group was significantly higher than that of FCP in AA genotype group (P=0.005). Conclusion A1168C polymorphism in PAX4 gene may not play an essential role in the genetic susceptibility of the islet autoantibody-negative KPD in Chinese Han population. However, A1168C variation may contribute to the predisposition to the male or <20 years old patients with islet autoantibody-negative KPD.

Identifying 1p LOH in Oligodendroglioma with Denaturing High Performance Liquid Chromatography / 中国康复理论与实践

@#Objective To set up the clinical examination method for 1p loss of heterozygosity (LOH).Methods Genomic DNA was isolated from tumor tissue and peripheral blood in oligodendroglioma patients. Denaturing high performance liquid chromatography (DHPLC) was used to examine the polymerase chain reaction (PCR) Results . 1p LOH was determined by the microsatellite sites.Results 1p LOH was identified by DHPLC Results .Conclusion DHPLC can clearly identify the 1p LOH with short time in oligodendroglioma patients.

Application of DHPLC for mutation detection of the fibrillin-1 in patients with Marfan syndrome / 中华检验医学杂志

Objective To detect fibrillin-1 gene (FBN1) mutations in patients with Marfan syndrome (MFS) by denaturing high-pedormance liquid chromatography (DHPLC) and DNA sequencing. Methods Genomic DNA was extracted from whole blood sample of 22 MFS patients. All 65 exens of FBN1 were amplified by polymerase chain reaction(PCR) respectively. Mutations were screened by DHPLC followed by DNA sequencing of the PCR products which showed different DHPLC profiles from the normals. Results Ten mutations of the FBN1 were found in 9 MFS patients. The mutations comprised four missense[5015G > C(C1672S),5309G > A(C1770Y),7241G > A(A2414G) and 7769G > A(C2590Y)], four nonsense [3295G > T ( E1099X ), 430"/insTCGT (G1441X), 4621C > T ( R1541X ) and 8080C > T (A2694X)], and two splice site mutations (IVS29 + 4A > T and IVSSO + 1G > A). Conclusion It is suggested that DHPLC coupled with DNA sequencing is an efficient method for the detection of FBN1 gene mutations, and it may be useful in diagnosis of MFS.

Detection of Escherichia coli O157:H7 by multiplex PCR-DHPLC / 中华微生物学和免疫学杂志

Objective To develop the PCR-denaturing high performance liquid chromatography (DHPLC) for detection of E. Coli O157: H7. Methods The virulence genes of Shiga-like toxin(SLT) and rfbE were specifically amplified by 2 sets of primers. The target gene fragments of the PCR assay were 224 bp and 499 bp, respectively. Results Analysis of 37 strains demonstrated that this PCR system was specific. The detection limit of the PCR was 4 CFU/ml. Conclusion These results indicated that the multiplex PCR-DHPLC assay can be used for specific and sensitive detection of E. Coli O157:H7.

Detection of Rifampin Resistant Mycobacterium tuberculosis complex using Denaturing HPLC / 대한진단검사의학회지

BACKGROUND: Tuberculosis (TB) remains an important cause of morbidity and mortality throughout the world. The surge of TB has been accompanied by an increase in multi-drug-resistant tuberculosis (MDR-TB). In this study, we developed a denaturing HPLC (DHPLC) method for detecting rpoB gene mutation as a rifampin resistance based on sequence. METHODS: In this study, we used 99 mycobacterial isolates grown in Ogawa media. At first, we used a PCR method that can amplify the 235 bp and 136 bp rpoB DNAs of Mycobacterium tuberculosis complex (MTB) and Non-tuberculous mycobacteria (NTM). And then, PCR-restriction fragment length polymorphism (RFLP) of rpoB DNA (342 bp), which comprises the Rif(T) region, was used for the differential identification of Mycobacteria. Finally, we detected these amplicons by DHPLC, compared to PCR-RFLP results, and performed sequencing. RESULTS: Among 99 mycobacterial isolates, 80 (81%) were MTB and 19 (19%) were NTM. NTM were identified to 7 different species by DHPLC and PCR-RFLP. rpoB mutation was detected in 9 (11%) of the MTB specimens. These results were confirmed by using sequencing. CONCLUSIONS: DHPLC provided a rapid, simple, and automatable performance for detection of rifampin resistant Mycobacterium tuberculosis complex and would be helpful as a supplemental method in high-throughput clinical laboratories.

Experimental study on rapid detection of lower respiratory tract pathogenic bacteria by denaturing high-performance liquid chromatography / 中华微生物学和免疫学杂志

Objective To construct a new molecular biological method for the analysis of microbial species in lower respiratory tract infections based on 16S rRNA gene by denaturing high-performance liquid chromatograph(DHPLC).Methods The universal primer set was analyzed basing on the highly conserved regions of 16S rRNA gene.DNA amplicons of lower respiratory tract were analyzed by DHPLC to generate peak profiles respectively.The incorporation of 40-bpGC clamp into the amplification primet was essential to effectively discriminate genetic differences identification.Results The primers could only amplify bacterial 16S rRNA.Bacterial of amplicons which incorporation of a 40-bpGC clamp were effectively discriminated genetic differences in DHPLC.The results of clinical isolares identification showed 100%according with the traditional method.Conclusion DHPLC has not only high accuracy,but also is a convenient,rapid and high-through technique for the discrimination bacteria.It has potential value in the detection of lower respiratory pathogenic bacteria.

Rapid Detection of Antibiotic Resistance of Klebsiella pneumoniae by Denaturing High-performance Liquid Chromatography (DHPLc) / 中华医院感染学杂志

OBJECTIVE To type the genes of plasmid DNA in 54 clinical Klebsiella pneumoniae isolates producing extended -spectrum beta-lactamases (SHV) by denaturing high-performance liquid chromatography (DHPLc) and evaluate their sensitivity and specificity, and explore a rapid and convenient method for detecting the antibiotic resistance of K. pneumoniae. METHODS Plasmid DNA from each extended-spectrum beta-lactamase (SHV) producing strain was subjected to PCR amplification. After we performed DNA sequencing of these amplicons and identification of mutation and their genotype, DHPLc was undertaken to investigate whether its results correlate the distinctive chromatogram with each genotype. RESULTS All the strains were found abnormal elution peaks (two or three peaks) which were different from each other. The result of DNA sequencing demonstrated that all the strains had DNA mutation in comparison with SHV-1. Moreover, DHPLc could produce specific peak patterns that correlate with genotype. CONCLUSIONS The sensitivity of DHPLc is 100% in this study. And each genotype is corresponded to specific peak pattern. So we can use DHPLc technique to type the genes of plasmid DNA in K. pneumoniae and detect mutations rapidly. DHPLc not only has high accuracy , but also is a convenient and rapid technique for the detection of mutation in the bacterial genome. It has a great potential clinical value.

Mutation Site of Cu/Zn superoxide dismutase in an amyotrophic lateral sclerosis family / 医学研究生学报

Objective: To identify the mutation points of Cu/Zn superoxide dismutase(SOD1) gene in an amyotrophic lateral sclerosis(ALS) family with a unique phenotype,and to compare the value of single strand conformation polymorphism(SSCP) and denaturing high performance liquid chromatography(DHPLC). Methods: Five exons of SOD1 gene were amplified by PCR. The difference of these products were analyzed by PCR-SSCP and DHPLC.DNA sequencing was used to examine the mutation. Results: ①Mutations were found in exons 2 and 5 in several family members.DNA sequencing revealed that a base pair insertion occurred in the codon area of exon 2 and in the non-codon area of exon 5.②The results of DHPLC tests proved double peaks in one member with ALS symptoms(Ⅲ1),which indicated the possibility of mutation in SOD1 exon 4.DNA sequencing revealed that there was a heterozygote,with a mutation of GAA to GGA in exon 4 in the member with double peak. Conclusion: ①The mutations in exons 2,4,5 were proved.Insertion of exon 2 may be responsible for the disease of the ALS family in Chongqing.②Compared with PCR-SSCP,DHPLC technique has been proven to be a rapid and reliable method for screening mutation site in large samples.

Denaturing high performance liquid chromatography for detection of point mutation of familial ALS / 第三军医大学学报

Objective To identify the point mutation of Cu/Zn superoxide dismutase(SOD1) gene in an amyotrophic lateral sclerosis(ALS) family and observe the value of denaturing high performance liquid chromatography(DHPLC). Methods DHPLC and DNA sequencing were used to examine SOD1 gene of the ALS family which had not been found mutation by PCR-SSCP. Results DHPLC tests proved double peaks in one member(Ⅲ_1), Which indicated the possibility of mutation in SOD1 exon 4. DNA sequencing revealed that there was a heterozygote,with mutation of GAA to GGA in exon 4, and with a substitution of glutacid by glycine. Conclusion As compared with PCR-SSCP, DHPLC technique has proved to be a rapid and reliable method for screening mutation site in large samples.