Detection of Methicillin-Resistant Staphylococcus aureus in Healthcare Workers Using Real-Time Polymerase Chain Reaction

Healthcare-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have recently become an important issue for healthcare facilities due to high rates of infection, mortality, and high treatment costs. We investigated the frequency of MRSA in healthcare workers (HCWs) via nasal carriage and assessed the performance of the LightCycler® MRSA Advanced test. We tested nasal swabs from the anterior nares of participating HCWs at an intensive care unit. Nasal swabs were identified as S. aureus, methicillin-sensitive or methicillin-resistant coagulase-negative staphylococci (MSCoNS or MRCoNS), or MRSA by using conventional culture and the LightCycler® MRSA Advanced test. Of the 142 HCWs who participated in this study, only 11 participants (7.8%) were MRSA-positive by conventional culture and MRSA ID, and 24 (16.9%) were positive for mecA by real time polymerase chain reaction (PCR). In terms of diagnostic performance, the LightCycler® MRSA Advanced test had a sensitivity of 100%, a specificity of 90.1%, a positive predictive value of 45.8%, and a negative predictive value of 100% compared with conventional culture method. The detection limit of the LightCycler® MRSA Advanced test was 10³ colony/mL. We concluded that real-time PCR was able to rapidly and sensitively detect MRSA in HCWs. However, MRSA must be confirmed by culture due to false positivity.     

Rapid genotyping of the human renin (REN) gene by the LightCycler® instrument: identification of unexpected nucleotide substitutions within the selected hybridization probe area

Preeclampsia is a serious disorder affecting nearly 3% of all in the Western world. It is associated with hypertension and proteinuria, and several lines of evidence suggest that the renin-angiotensin system (RAS) may be involved in the development of hypertension at different stages of a preeclamptic pregnancy. In this study, we developed rapid genotyping assays on the LightCycler® instrument to allow the detection of genetic variants in the renin gene (REN) that may predispose to preeclampsia. The method is based on real-time PCR and allele-specific hybridization probes, followed by fluorescent melting curve analysis to expose a change in melting temperature (T_m). Ninety-two mother-father-child triads (n=276) from preeclamptic pregnancies were genotyped for three haplotype-tagging single nucleotide polymorphisms (htSNPs) in REN. All three htSNPs (rs5705, rs1464816 and rs3795575) were successfully genotyped. Furthermore, two unexpected nucleotide substitutions (rs11571084 and rs61757041) were identified within the selected hybridization probe area of rs1464816 and rs3795575 due to aberrant melting peaks. In conclusion, genotyping on the LightCycler® instrument proved to be rapid and highly reproducible. The ability to uncover additional nucleotide substitutions is particularly important in that it allows the identification of potentially etiological variants that might otherwise be overlooked by other genotyping methods.     

The rs8099917 polymorphism, when determined by a suitable genotyping method, is a better predictor for response to pegylated alpha interferon/ribavirin therapy in Japanese patients than other single nucleotide polymorphisms associated with interleukin-28B

We focused on determining the most accurate and convenient genotyping methods and most appropriate single nucleotide polymorphism (SNP) among four such polymorphisms associated with interleukin-28B (IL-28B) in order to design tailor-made therapy for patients with chronic hepatitis C virus (HCV) patients. First, five different methods (direct sequencing, high-resolution melting analysis [HRM], hybridization probe [HP], the InvaderPlus assay [Invader], and the TaqMan SNP genotyping assay [TaqMan]) were developed for genotyping four SNPs (rs11881222, rs8103142, rs8099917, and rs12979860) associated with IL-28B, and their accuracies were compared for 292 Japanese patients. Next, the four SNPs associated with IL-28B were genotyped by Invader for 416 additional Japanese patients, and the response to pegylated interferon/ribavirin (PEG-IFN/RBV) treatment was evaluated when the four SNPs were not in linkage disequilibrium (LD). HRM failed to genotype one of the four SNPs in five patients. In 2 of 287 patients, the results of genotyping rs8099917 by direct sequencing differed from the results of the other three methods. The HP, TaqMan, and Invader methods were accurate for determination of the SNPs associated with IL-28B. In 10 of the 708 (1.4%) patients, the four SNPs were not in LD. Eight of nine (88.9%) patients whose rs8099917 was homozygous for the major allele were virological responders, even though one or more of the other SNPs were heterozygous. The HP, TaqMan, and Invader methods were suitable to determine the SNPs associated with IL-28B. The rs8099917 polymorphism should be the best predictor for the response to the PEG-IFN/RBV treatment among Japanese chronic hepatitis C patients.     

高分辨熔解曲线分析技术检测中国人常见β地中海贫血基因突变

目的应用高分辨熔解曲线分析技术检测中国人常见β地中海贫血基因突变。方法 PCR扩增HBB基因3个外显子、外显子-内含子交界区及第2内含子部分区域,采用LightScanner对PCR产物进行高分辨熔解曲线分析,通过DNA;;测序对HRM结果进行验证。结果对48例正常对照的HRM分析显示,在HBB基因的突变筛查区域存在两个常见SNPs;在验证实验中,对37例已知基因型患者的HRM分析显示,所有突变均得到正确检测且每种基因型都有其独特的熔解曲线;在双盲实验中,对70例未知样本的HRM分析共检出28个异常熔解曲线,与测序结果完全一致,HRM检测的灵敏度和特异性均为100%。结论高分辨熔解曲线分析法检测HBB基因突变具有简单快速、高效敏感、成本低廉等优点,不仅可用于β地贫的突变筛查,还可对已知突变进行基因分型。     

Real-time polymerase chain reaction assay based on high-resolution melting analysis for the determination of the rs12979860 polymorphism involved in hepatitis C treatment response

Treatment of chronic hepatitis C is associated with varying success rates, substantial medical costs and serious side effects. Several host polymorphisms have been identified near the IL28B gene, of which the homozygous rs12979860 CC was found to be associated with significantly favourable treatment outcome. To determine accurately the presence of this variant, a real-time PCR assay with detection based on post-PCR high-resolution melting analysis (HRM) was developed. The assay, performed on a Roche LightCycler 480, was able to differentiate among all three rs12979860 variants (CC, TT, CT) across a dynamic range of four orders of magnitude (10³-10⁷). The sensitivity of the assay was determined at a 95% detection level to be 44.6 copies/reaction for the CC variant, 57.1 copies/reaction for the TT variant and 47.4 copies/reaction for the CT variant. Input DNA amount above 10³ copies/reaction is recommended for clinical samples to ensure optimal performance. Clinical performance was assessed on 60 clinical samples by comparative testing using the assay and Sanger sequencing. Concordant results were obtained for all 60 samples, showing high specificity of the assay. The novel assay can be easily added to the testing repertoire of virological laboratories, providing additional information for physicians treating chronic hepatitis C patients.     

High-resolution melting combines with Bayes discriminant analysis: a novel hepatitis C virus genotyping method

Current hepatitis C virus (HCV) genotyping techniques are often highly technical, costly, or need improvements in sensitivity and specificity. These limitations indicate the need of novel methods for HCV genotyping. The present study aimed to develop a novel genotyping method combining high-resolution melting (HRM) analysis with Bayes discriminant analysis (BDA). Target gene fragment including 5′-untranslated and core region was selected. Four or five inner amplicons for every serum were amplified using nested PCR, HRM was used to determine the melting temperature of the amplicons, and HCV genotypes were then analyzed utilizing BDA. In initial genotyping (HCV genotypes were classified into 1b, 2a, 3a, 3b, and 6a), both the overall accuracy rate and the cross-validation accuracy rate were 92.6 %, external validation accuracy rate was 95.0 %. To enhance the accuracy rate of genotyping, HCV genotypes were firstly classified into 1b, 3a, 3b, and 2a–6a, followed by a supplementary genotyping for 2a–6a. Both the overall accuracy rate and the cross-validation accuracy rate reached 97.5 %, and external validation accuracy rate was 100 %. Comparing adjusted HRM genotyping with type-specific probe technique, the difference in accuracy rates was not significant. However, the limit of detection and cost were lower for HRM. Comparing with sequencing, the limit detection of HRM was the same as the former, but the cost of HRM was lower. Hence, HRM combined with BDA was a novel method that equipped with superior accuracy, high sensitivity, and lower cost and therefore could be a better technique for HCV genotyping.     

A duplex probe-directed recombinase amplification assay for detection of single nucleotide polymorphisms on 8q24 associated with prostate cancer

Single nucleotide polymorphisms (SNPs) have important application value in the research of population genetics, hereditary diseases, tumors, and drug development. Conventional methods for detecting SNPs are typically based on PCR or DNA sequencing, which is time-consuming, costly, and requires complex instrumentation. In this study, we present a duplex probe-directed recombinase amplification (duplex-PDRA) assay that can perform real-time detection of two SNPs (rs6983267 and rs1447295) in four reactions in two tubes at 39°C within 30 min. The sensitivity of duplex-PDRA was 2×10³-10⁴ copies per reaction and no cross-reactivity was observed. A total of 382 clinical samples (179 prostate cancer patients and 203 controls) from northern China were collected and tested by duplex-PDRA assay and direct sequencing. The genotyping results were completely identical. In addition, the association analysis of two SNPs with prostate cancer risk and bone metastasis was conducted. We found that the TT genotype of rs6983267 (OR: 0.42; 95%CI: 0.23-0.78; P=0.005) decreased the risk of prostate cancer, while the CA genotype of rs1447295 (OR: 1.89; 95%CI: 1.20-2.96; P=0.005) increased the risk of prostate cancer. However, no association between the two SNPs (rs6983267 and rs1447295) and bone metastasis in prostate cancer was found in this study (P>0.05). In conclusion, the duplex-PDRA assay is an effective method for the simultaneous detection of two SNPs and shows great potential for widespread use in research and clinical settings.     

胃动素基因多态性与先天性肥厚性幽门狭窄的相关性

目的 探讨中国汉族人群中胃动素基因多态性位点rs2281820[44C＞T]与先天性肥厚性幽门狭窄(CHPS)发病的关系.方法 采用病例-对照研究的方法,在中国汉族人群中收集2006年7月至2009年8月在本院就诊的29例CHPS散发病例为CHPS组,60例无血缘关系的健康成人为对照组.采用PCR及测序的方法进行基因分型,比较2组间基因型及等位基因分布.结果 与对照组比较,CHPS组的CC基因型频率及C等位基因频率差异无统计学意义[86.21%(25/29)比80.00%(48/60),91.38%(53/58)比89.17%(107/120),均P＞0.05].结论 胃动素基因的rs2281820[44C＞T]位点多态性与中国汉族人群CHPS发病无相关.     

IL-10 基因多态性与溃疡性结肠炎易感性的关系及对临床预后的影响

目的:探讨IL-10 多态位点的基因多态性与溃疡性结肠炎的易感性及对临床预后的影响。方法:采用病例对照研究设计,选取80例溃疡性结肠炎患者作为病例组,另外选性别和年龄匹配的健康受试者作为对照组。所有患者治疗前抽取空腹静脉血并提取DNA,设计819 T/ C(rs1800871)、592A/C(rs1800872)、 -1082 G/ A(rs1800896)PCR 引物进行PCR 扩增,扩增产物酶切后进行琼脂糖凝胶电泳以确定基因类型,采用Logistic 回归计算校正相对危险度(OR)和95% 置信区间(95%CI)评价基因多态性与溃疡性结肠炎的易感性,并分析对临床预后的影响。结果:(1) 病例组患者IL鄄10 多态位点rs1800896 基因类型AA、GG 和AG 分布频率与对照组受试者差异具有统计学意义(P<0.01);(2)与rs1800896 基因型AA 比较,基因型为GG 的患者溃疡性结肠炎危险性显著升高(P<0.01),并且临床缓解率显著降低(P<0.01);(3)病例组患者IL-10多态位点rs1800871 基因类型CC、CT 和TT 分布频率与对照组受试者差异无统计学意义(P>0.05);(4)病例组患者IL鄄10 多态位点rs1800872 基因类型AA、AC 和CC 分布频率与对照组受试者差异无统计学意义(P>0.05)。结论:IL-10 多态位点rs1800896 基因类型GG 可增加溃疡性结肠炎的易感性,并且显著降低患者的临床预后。     

A straightforward genotyping of the relevant IL28B SNPs for the prediction of hepatitis C treatment outcome

A sustained virological response is not achieved by a significant proportion of chronic hepatitis C patients treated with interferon-based regimens. Due to the associated side effects and high costs, therapy response markers have been thoroughly sought. Two Single Nucleotide Polymorphisms (SNPs), rs12979860 and rs8099917, which are located upstream from the IL28B gene, have been remarkably described to have a strong association with treatment efficacy. The aim of this study was to develop a straightforward method for genotyping such polymorphisms. A Polymerase Chain Reaction (PCR) followed by enzymatic restriction of amplicons was established for SNPs genotyping. Online computation resources were employed for retrieving reference sequences, such as the selection of oligonucleotides and restriction enzymes. Two pairs of primers were designed and validated for the amplification of segments encompassing rs12979860 (694bp) and rs8099917 (496bp) with common thermocycling parameters. The endonucleases Hpy166II and BsrDI were selected and used for allelic discrimination related to rs12979860 (C/T) and rs8099917 (T/G), respectively. The expected electropherotypes were confirmed for all possible genotypes in 75 blood samples. In addition, the results were validated by sequencing. The method constitutes a simple and reliable assay, which may be readily available for genotyping of rs12979860 and rs8099917 in laboratories that support hepatitis C treatment centers.     

高分辨熔解曲线技术鉴定多药耐药基因外显子单核苷酸多态性

目的高分辨熔解曲线技术（HRM）检测多药耐药基因（MDR1）外显子12单核苷酸多态性（SNP）。方法采用高分辨熔解技术对MDR1基因外显子12的SNP C1236T位点进行基因分型,以其-401C〉T位点为例设计PCR扩增引物,按PCR扩增效率和熔解曲线进行退火温度、升温速度等条件的优化,并用此优化体系基因分型20例外周血标本,以测序验证。结果 20例标本经测序与检测结果一致。结论高分辨熔解曲线技术检测SNP是一种低成本、简便易行、常规化,高通量的基因分型方法,能用于大规模临床筛查。     

Genomic DNA pooling for whole-genome association scans in complex disease: empirical demonstration of efficacy in rheumatoid arthritis

A pragmatic approach that balances the benefit of a whole-genome association (WGA) experiment against the cost of individual genotyping is to use pooled genomic DNA samples. We aimed to determine the feasibility of this approach in a WGA scan in rheumatoid arthritis (RA) using the validated human leucocyte antigen (HLA) and PTPN22 associations as test loci. A total of 203 269 single-nucleotide polymorphisms (SNPs) on the Affymetrix 100K GeneChip and Illumina Infinium microarrays were examined. A new approach to the estimation of allele frequencies from Affymetrix hybridization intensities was developed involving weighting for quality signals from the probe quartets. SNPs were ranked by z-scores, combined from United Kingdom and New Zealand case-control cohorts. Within a 1.7 Mb HLA region, 33 of the 257 SNPs and at PTPN22, 21 of the 45 SNPs, were ranked within the top 100 associated SNPs genome wide. Within PTPN22, individual genotyping of SNP rs1343125 within MAGI3 confirmed association and provided some evidence for association independent of the PTPN22 620W variant (P=0.03). Our results emphasize the feasibility of using genomic DNA pooling for the detection of association with complex disease susceptibility alleles. The results also underscore the importance of the HLA and PTPN22 loci in RA aetiology.     

小扩增子基因分型方法在检测XRCC1 Arg194Trp多态性分析中的应用

目的 应用小扩增子基因分型方法检测正常人宫颈癌易感基因XRCC1 Arg194Trp位点多态性改变.方法 提取10例健康人外周血基因组DNA;PCR扩增XRCC1 基因 Arg194Trp位点,扩增产物进行小扩增子基因分型分析.并应用测序方法对分析结果进行验证.结果 HRM检测结果分别显示3种基因型:XRCC1 Arg194Arg(C/C)、XRCC1 Arg194Trp(C/T)和XRCC1 Trp194Trp(T/T).测序结果与小扩增子基因分型方法分析结果完全一致.结论 应用小扩增子基因分型方法可以对XRCC1 Arg194Trp进行准确分型.     

Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow

AimTo evaluate critical steps in Illumina® Human mtDNA Genome assay: target enrichment, limited-cycle PCR, and library normalization, in order to optimize the protocol for analysis of whole mitochondrial genomes from human reference samples.MethodsThree long-range high-fidelity DNA polymerases (Platinum^TM PCR SuperMix High Fidelity, LA Taq® Hot Start, and PrimeSTAR® GXL) were tested for their performance in the amplification of mtDNA fragments. Sequencing results of ten samples, as well as negative controls, which underwent library preparation with 12 and 15 cycles in limited-cycle PCR were compared. Additionally, two library normalization methods were compared: bead-based normalization vs quantification and individual normalization.ResultsPrimeSTAR® GXL performed best for mitochondrial DNA enrichment. Increment of amplification cycles to 15 in limited-cycle PCR step did not affect either the sequencing process or variant calling. Library quantification combined with individual library-by-library dilution outperformed bead-based normalization.ConclusionOptimizations described herein provide beneficial insights for laboratories aiming at implementation and/or advancement of similar massively parallel sequencing workflows (eg, small genomes, PCR amplicons, and plasmids).

Library preparation in massively parallel sequencing (MPS) protocols is a sensitive process, usually consisting of multiple elaborate steps. To ensure high quality of sequencing results, it is important to optimize library preparation according to characteristics of a particular target molecule. There are several critical aspects of Illumina® Human mtDNA Genome (1) assay for analysis of whole mitochondrial DNA (mtDNA) on MiSeq® instrument: initial enrichment of the target molecule (achieved, in this case, by long-range PCR); PCR step wherein index-adapter oligonucleotides are added and libraries are amplified (termed “limited-cycle PCR” step); and normalization of libraries prior to their pooling for sequencing.In this study, we aimed to test three long-range high-fidelity DNA polymerases for their performance in amplification of mtDNA fragments, in order to determine the one best suited for Illumina® assay, in which mitochondrial genomes are amplified in two large fragments (sizes 9.1 kb and 11.2 kb) (Supplementary Figure 1(Supplementary Figure 1)).Various optimizations and evaluations have been previously published (2-6), but, to our knowledge, none of them assessed the impact of increasing the number of amplification cycles in limited-cycle PCR. Therefore, we also aimed to test and observe how sequencing results were affected by this step and whether there were any adverse effects that would impact variant calling.Lastly, we aimed to compare two library normalization methods: bead-based normalization vs quantification and individual normalization. Nextera® XT Library Prep Kit (Illumina, San Diego, CA, USA) has been known to produce uneven read depth profiles (2-4,6-8). Therefore, a great risk in this protocol is potential loss of sequence information in regions that achieve very low read depth (ie, too low for analysis and subsequent variant calling), or possibly receive no reads at all. The choice of library normalization method may affect this through distribution of reads among the sequenced libraries. So, a method that provides more uniform distribution of reads would be preferable, which we hypothesized would be the method of quantification and individual normalization rather than bead-based normalization.     

小扩增子基因分型方法在检测XRCC1 Arg194Trp多态性分析中的应用

目的 应用小扩增子基因分型方法检测正常人宫颈癌易感基因XRCC1 Arg194Trp位点多态性改变.方法 提取10例健康人外周血基因组DNA;PCR扩增XRCC1 基因 Arg194Trp位点,扩增产物进行小扩增子基因分型分析.并应用测序方法对分析结果进行验证.结果 HRM检测结果分别显示3种基因型:XRCC1 Arg194Arg(C/C)、XRCC1 Arg194Trp(C/T)和XRCC1 Trp194Trp(T/T).测序结果与小扩增子基因分型方法分析结果完全一致.结论 应用小扩增子基因分型方法可以对XRCC1 Arg194Trp进行准确分型.     

小扩增子基因分型方法在检测XRCC1 Arg194Trp多态性分析中的应用

目的 应用小扩增子基因分型方法检测正常人宫颈癌易感基因XRCC1 Arg194Trp位点多态性改变.方法 提取10例健康人外周血基因组DNA;PCR扩增XRCC1 基因 Arg194Trp位点,扩增产物进行小扩增子基因分型分析.并应用测序方法对分析结果进行验证.结果 HRM检测结果分别显示3种基因型:XRCC1 Arg194Arg(C/C)、XRCC1 Arg194Trp(C/T)和XRCC1 Trp194Trp(T/T).测序结果与小扩增子基因分型方法分析结果完全一致.结论 应用小扩增子基因分型方法可以对XRCC1 Arg194Trp进行准确分型.     

Identification of polymorphisms in the XIAP gene and analysis of association with lung cancer risk in a Korean population

The X-linked inhibitor of apoptosis protein (XIAP) is a potent mammalian IAP, and has been shown to play an important role in development and progression of cancer. Polymorphisms in the XIAP gene may influence XIAP production or activity, thereby modulating susceptibility to lung cancer. To test this hypothesis, we first screened for polymorphisms in the XIAP gene by direct sequencing of genomic DNA samples from 27 healthy Korean women and then performed a case-control study to evaluate the association between the polymorphisms and the risk of lung cancer. The XIAP genotypes were determined by polymerase chain reaction amplification and melting curve analysis in 582 lung cancer patients and in 582 healthy control subjects who were frequency-matched for age and sex. We identified 12 single nucleotide polymorphisms (SNPs), one novel SNP [30051C>G (A321G) in exon 3] and the following 11 known SNPs: 192G>C (rs5956578), 262C>T (rs28382699), 318C>T (rs5958318), and 374C>T (rs12687176) in the putative promoter; 26615A>G (rs2355676) in intron 1; 41725A>G (rs5958338) in intron 5; 42009A>C (Q423P, rs5956583) in exon 6; 48162T>C (rs17334739) and 48228C>T (rs28382739) in intron 6; and 48542A>G (rs28382740) and 49333G>T (rs28382742) in 3'-UTR. Four of these 12 SNPs were selected for large-scale genotyping based on their frequencies and haplotype tagging status: 262C>T, 318C>T, 374C>T, and 42009A>C. The four XIAP polymorphisms and their haplotypes exhibited no apparent relationship with the risk of lung cancer. In addition, we observed no evidence of effect modification by age, sex, smoking history, or tumor histology. These results suggest that XIAP polymorphisms do not significantly affect susceptibility to lung cancer in Koreans.     

E6-E7 based nested multiplex PCR assay for genital HPV detection and simultaneous typing of 15 high and low-risk HPV types

PurposeDue to a wide range of Human Papillomavirus (HPV) types associated with genital cancers; HPV genotyping remains important for the introduction of an appropriate vaccine, disease diagnosis, follow-up and epidemiological surveys. Currently, available molecular genotyping assays are not only expensive but also requires dedicated and expensive equipment which is not feasible in the majority of low-and-middle-socioeconomic countries. The purpose of the study was to develop and evaluated a cost-effective nested-multiplex polymerase chain reaction (NM-PCR) assay for HPV genotyping.MethodsHPV-DNA containing plasmids and cervical scrapings from histologically confirmed cervical cancer cases were used to evaluate the NM-PCR. In the first round PCR, a set of consensus primers were used to amplify 38 mucosal HPV types. HPV Type-specific primers were used in the second-round polymerase chain reaction (PCR) to amplify 15 HPV types in three multiplex cocktails. The assay sensitivity was determined with the control panel containing one to 10¹⁰genome equivalents (GE). DNA sequencing was done to confirm the PCR results.ResultsThe assay was able to amplify all HPV types and detected as few as 50GE per reaction. A total of 23 endo-cervical samples obtained from healthy, HPV negative subjects and 52 histologically confirmed cervical scrapings were processed for HPV genotyping by NM-PCR. HPV DNA was detected in all histologically confirmed samples. DNA sequencing results showed complete concordance with PCR results.ConclusionsThe designed nested PCR based assay had good concordance with clinical histology and sequencing results and appears to be a promising tool for HPV genotyping especially in resource-constrained settings.     

Evaluation of 14 Y-chromosomal Short Tandem Repeat Haplotype with Focus on DYS449, DYS456, and DYS458: Czech Population Sample

Aim

To evaluate the novel triplex polymerase chain reaction (PCR) assay for the analysis of polymorphic Y-chromosomal short tandem repeat loci (Y-STR).

Methods

A total of 14 Y-STR loci was analyzed. Allele frequencies for 3 tetrameric Y-STR loci (DYS449, DYS456, and DYS458) and extended haplotype loci typed by Y-PLEX^TM 12 system were investigated in a sample of 50 unrelated healthy Czech male donors. We computed the relevant intra-population statistic parameters for our data (gene diversity, average gene diversity over loci, and mean number of pairwise differences) and compared our sample set with other Central European populations using R_ST pairwise genetic distance.

Results

We focused on the comparison of genetic diversity between the Y-STR extended haplotype loci and that of the 3 additional loci, and on the benefit of using DYS449, DYS456, and DYS458 in forensic and population genetics applications. Total gene diversity in our sample set was 0.998367 when using all 14 loci. Our data analysis revealed very high genetic diversity at DYS449 locus (0.876735), which surpasses even the diversity at DYS385a/b (0.819592). Population comparison showed no difference between Czech, Bavarian, Austrian, and Saxon sample set. A minor difference was found between Czech and Polish sample set.

Conclusion

Typing of 3 Y-chromosomal microsatellite polymorphisms may provide a useful complement to already established sets of Y-STRs.DNA typing using a number of polymorphic short tandem repeats on human Y chromosome (Y-STR) has already become a broadly applied approach in areas such as forensic genetics and paternity testing (1). Also, the possibility of amplification of multiple STRs in a single polymerase chain reaction (PCR) provides a very efficient and reliable genotyping tool. Until recently, 219 Y-STRs have been described (2), most of which are polymorphic. In forensic genetics applications, Y-STRs are useful for discrimination of paternal lineages rather than for individual identification. In combination with the biallelic polymorphisms, Y-STRs are also applied in population genetic studies.The main aim of this study was to design a triplex PCR assay that allows fragmentation analysis of samples labeled with only one fluorescent dye. The loci DYS449, DYS456, and DYS458 were chosen for their reported high diversity in Euro-American population (3), as well as for their absence in the broadly used commercial forensic kits (PowerPlex® Y System [Promega, Madison, WI, USA], Mentype® Argus Y-12QS [Biotype, Dresden, Germany]), although DYS456 and DYS458 (not DYS449) are included in widely used AmpFℓSTR® Yfiler® PCR Amplification Kit (Applied Biosystem, Foster City, CA, USA) (4). DYS449 and DYS456 have also been used, together with other 25 Y-STR loci, in a major population study (5). Here we report on allele frequency data and basic intra-population diversity indices of the 3 Y-STRs, as well as those of 11 other Y-STR loci included in the extended haplotype set that were analyzed in the Czech population sample.     

CYP2C19、CYP3A5基因多态性与心肌梗死的相关性研究

目的探讨CYP2C19、CYP3A5基因的多态性与心肌梗死发病风险的相关性。方法随机选取心肌梗死患者及健康对照各500例,采用荧光PCR法和Sanger测序分别检测其CYP2C19、CYP3A5基因的多态性,用Logistic回归分析其与心肌梗死的相关性,用Quanto软件评估统计学效能。结果CYP2C19基因rs4986893位点的AG、GG基因型和A等位基因的频率以及CYP3A5基因rs776746位点的AA、AG、GG基因型和G等位基因频率在两组之间的差异具有统计学意义(P<0.05),CYP2C19基因rs4244285、rs12248560位点的基因型和等位基因以及rs4986893位点的AA基因型的频率在两组之间差异无统计学意义(P>0.05)。在校正年龄、性别、体质指数后,Logistic回归分析显示CYP2C19基因rs4986893的AG基因型和A等位基因以及CYP3A5基因rs776746的GG基因型和G等位基因可能是心肌梗死发病的风险因素,而rs4986893的GG基因型以及rs776746的AA、AG基因型可能是心肌梗死的保护因素。依据样本量、样本结构和等位基因频率以及Quanto分析,本研究的结果具有理想的统计学效能(99%)。结论CYP2C19、CYP3A5基因的多态性可能增加心肌梗死的发病风险。