应用SNPstream技术检测MASP2基因的多态性

目的：应用一种高通量单核苷酸多态性（SNP）检测方法——SNPstream技术检测甘露聚糖结合凝集素相关丝氨酸蛋白酶-2（MASP2）基因的多态性。方法：收集北京汉族人群SARS病例96例和正常对照96例,用SNPstream技术检测样本的MASP2基因多态性,并用PCR产物直接测序技术对其中一个位点rs2273346进行分型,以验证SNPstream技术的准确性。结果：192例样本的MASP2基因rs2273346位点SNPstream技术分型结果与测序结果完全相符,2种方法的基因型分型结果具有很好的一致性。结论：SNPstream技术是高通量SNP检测的良好工具,准确性高,所需样本量低,在大规模人群SNP筛检中具有良好的发展前景。     

适用于降解样本的201个遗传标记检测体系的构建和验证

近年来,法医实践中复杂案件数量逐渐增多,需要联合使用短串联重复序列(short tandem repeat,STR)、单核苷酸多态性(single nu cleotide p olymorphis ms,SNP)、插入缺失多态性(insert ion/deletion p olymorphism,InDel)、微单倍型(microhaplotype,MH)等不同类型的遗传标记,为案件提供更多的参考信息。本研究筛选了24个常染色体STR(autosomes STR,A-STR)、24个Y染色体STR(Y-STR)、110个A-SNP、24个Y-SNP、9个A-InDel、1个Y-InDel、8个MH和Amelo genin共201个遗传标记,建立二代测序检测体系HID_AM Panel v1.0。根据DNA分析方法科学工作组(Scientific Working Group on DNA Analy sis M ethods,SWGDAM)的验证指南,对该体系的重复性、准确性、灵敏度、对降解样本的适用性、物种特异性、抗抑制性等指标进行评估。本体系分型结果与基于毛细管电...     

基于微滴式数字PCR检测肠癌病人游离环状DNA KRAS突变的新方法

基于微滴式数字聚合酶链式反应(Droplet digital polymerase chain reaction,dd PCR)设计一种检测肠癌游离循环DNA(Circulating cell free DNA,cf DNA)中KRAS(V-Ki-ras2 Kirsten ratsarcoma viral oncogene homolog)基因突变的新方法并评估其灵敏度和准确性。根据肠癌病人KRAS基因的突变类型设计并合成,采用dd PCR扩增并评估其灵敏度和准确性;根据AMRS-PCR引物设计原理设计KRAS基因的实时定量PCR扩增引物并评估其准确性,进而比较dd PCR和q PCR二者之间的优缺点;最后针对52例肠癌病人的cf DNA采用dd PCR进行检测,研究dd PCR在cf DNA KRAS基因突变检测的应用。成功使用dd PCR和q PCR两种方法对KRAS野生型及7种突变型建立检测方法,使用质粒标准品及实际样品验证该两种方法可行并对其假阳性率、线性范围及检测下限等性能进行了评价,最后成功对52例临床患者和20例正常人的血浆cf DNA样本进行检测,临床灵敏度为97.64%,临床特异性为81.43%。dd PCR的检测性能优于q PCR,LOD达到个位数DNA拷贝,最低可确认突变浓度达到0.01%–0.04%。样本提取效率在方法学建立中也十分重要,直接影响到灵敏度和Cut Off值的判定。临床患者检测结果显示其KRAS突变率接近报道水平。     

畜禽养殖粪污中典型致病菌的三重微滴式数字PCR定量检测方法的建立北大核心CSCD

为精准、快速检测畜禽养殖粪污中典型致病微生物,减少人畜共患病的传播风险,以金黄色葡萄球菌、大肠埃希氏菌(O157:H7)和肠炎沙门氏菌3种常见致病菌为研究对象,通过筛选其特异性引物与探针、优化反应系统,建立起快速、稳定的多重微滴式数字PCR(droplet digital PCR,ddPCR)反应体系。通过检测不同菌株验证该体系的特异性,并确定畜禽养殖废弃物致病菌的检出限,开发出多重微滴数字PCR快速检测方法。研究结果表明,各对引物探针对目标菌株均能扩增,ddPCR体系内未出现交叉反应,检测肠炎沙门氏菌的绝对定量检测低限为0.68 copies/μL;检测金黄色葡萄球菌的绝对定量检测低限为0.79 copies/μL;检测大肠埃希氏菌的绝对定量检测低限为1.02 copies/μL。研究建立的方法可实现对畜禽养殖粪污中3种典型致病菌的高效率、高精度的检测。     

基于微流控芯片的InDel快速族群推断体系研究

目的 QuickTargSeq全集成法医DNA现场快速检测系统是国内首台自主研制的现场快检仪,可应用于InDel族群推断检测,2 h左右完成“样本进-结果出”的快速自动化InDel分型。本文对InDel族群推断微流控芯片检测体系的性能进行评估,以期为实践应用提供参考。方法 使用InDel族群推断微流控芯片检测体系,对体系的灵敏度、干扰物耐受性、成功率、分型准确率、精确性、准确性、峰平衡性及检材适应性进行验证评估,同时对测试样本的族群来源进行推断。结果 138份样本的全集成检测成功率为95.65%,分型准确率为98.85%;DNA模板量≥5 ng时,可获得完整InDel分型,口腔拭子样本最佳采集次数为口腔内壁左右两侧各刮擦8次,血卡样本最佳检测方式为6片（Φ=2 mm）;所有基因座的平均杂合子峰高比值为0.86;10次运行的等位基因分型标准物（allelic ladder）片段大小标准差均在0.3 bp以内,测试样本等位基因和相应的等位基因分型标准物之间的片段准确性均在0.5 bp以内。结论 该体系可实现对口腔拭子、血卡、唾液卡及烟蒂样本的准确分型,能够准确推断样本的族群来源。     

Y染色体短串联重复序列微流控芯片复合扩增检测体系研究

目的 构建Y染色体短串联重复序列（Y-STR）微流控芯片扩增检测试剂,并进行性能验证,实现Y-STR基因座的快速全集成检测。方法 使用Y-STR微流控芯片检测体系,对其灵敏度、成功率和分型准确率、峰平衡性、精准性和准确性、检材适应性、混合物检测能力和抗抑制性进行验证评估。结果 DNA标准品9948的模板量≥8 ng,血卡片数≥3片以及口腔拭子刮擦次数≥7次时可获得Y-STR完整分型;165份样本的全集成检测成功率为91.52%,分型准确率为99.74%;不同荧光通道之间的峰高比值为89.81%;10次运行的等位基因分型标准品的片段大小标准差均在0.5 bp以内,20份样本全集成检测的等位基因片段和相应的等位基因标准品之间的片段准确性均在0.5 bp以内;能够对口腔拭子、血卡、唾液卡、烟蒂、血棉签、布片精斑等检材进行准确分型;混合样本中较小贡献者与较大贡献者在1∶3的比例时可获得完整基因分型;在不同浓度的腐殖酸（50～400 mg/L）、靛蓝（20～100 nmol/L）、血红蛋白（100～500μmol/L）等抑制物的干扰下,该体系可获得完整基因分型。结论 该体系可应用于国产Quick...     

人博卡病毒TaqMan探针实时定量PCR检测方法的建立及初步应用

目的:建立人博卡病毒(HBoV)核酸特异、快速、敏感的TaqMan探针实时定量PCR检测方法,并对临床样本进行检测。方法:比对编码HBoV非结构蛋白NP-1的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量PCR检测方法,并与传统PCR方法进行比较,然后分别对两者的灵敏性、特异性、稳定性及临床样本检验的适用性等进行评价。结果:所建立的实时定量PCR检测方法可用于HBoV的特异性检测;相对于传统PCR所达到的250拷贝/反应的检测灵敏度,实时定量PCR的检测灵敏度可高达10拷贝/反应,检测范围为109～101拷贝/反应,且具有良好的特异性和重复性;初步用于76份临床呼吸道标本检测,检出阳性5例,高于普通PCR方法(3/76)。结论:建立了HBoV TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,为开展HBoV流行病学监测及早期临床诊断提供了技术手段。     

双重数字PCR在转基因大豆检测中的应用

利用微滴式数字PCR(droplet digital PCR, ddPCR)平台建立针对MON87705、MON87769、DP356043三种转基因大豆中外源基因的双重PCR检测方法。利用双重数字PCR方法检测特异性、定量范围等参数,优化所用引物探针组合及实验体系程序,检测外源基因与内标准基因的拷贝数。结果表明,所用引物探针组合在数字PCR方法中仅对目标大豆品系有荧光信号,具有特异性,可用于转基因大豆品系的筛选与鉴别。检测了大豆的转基因成分含量,结果与材料标准品参数基本一致,并根据结果设定定量检测限为0.5%,定性检测限为0.05%,可满足低纯度样品检测的需求。双重数字PCR体系能够准确且稳定的满足实际检测需要,在实际应用上具有良好的发展前景。     

转基因玉米2A-5特异性PCR方法的建立

根据转基因玉米2A-5的旁侧序列信息,设计并筛选出最佳特异性引物及Taqman探针组合2A-5-5-QF8、2A-5-5-QR8、2A-5-5-QP8,优化了该引物探针组合的反应体系,建立了转基因玉米2A-5转化体特异性PCR检测方法。将特异性引物及Taqman探针组合用于qRT-PCR和ddPCR技术,研究了转基因玉米2A-5转化体的定量检测方法,发现PCR定量检测转基因含量与测定样品转基因含量之间呈高度正相关。研究获得的转基因玉米2A-5转化体特异性PCR检测方法及定量qRT-PCR、ddPCR检测方法具有较高的特异性、准确性和灵敏度,是今后准确、高效检测2A-5及其产品的有效方法之一,同时也为我国转基因生物安全监管提供了良好的技术支撑。     

双歧杆菌属特异性定量引物的设计及验证

【目的】旨在设计一对双歧杆菌属特异性引物以检测不同样品中低丰度双歧杆菌的含量。【方法】在NCBI中下载57株双歧杆菌全基因组序列,以其共有单拷贝核心基因为目的片段设计双歧杆菌属特异性引物;并对引物进行PCR初筛和特异性复筛;之后借助ddPCR(Droplet Digital PCR,微滴式数字PCR)依次对筛选出的引物进行特异性、灵敏度和实用性验证。【结果】引物Bif-D-9特异性最好,可扩增出4株双歧杆菌而不能扩增20株非双歧杆菌中的任何一株菌;同时通过ddPCR仪定量稀释后的DNA,其扩增结果呈线性下降趋势,证明其灵敏度较好;另外,Bif-D-9结合ddPCR定量出婴儿粪便中双歧杆菌的拷贝数为71 copies/μL,母亲粪便中双歧杆菌的拷贝数为2.7 copies/μL,证明了该方法的实用性。【结论】引物Bif-D-9具有双歧杆菌属特异性,且灵敏度较高、实用性较好,适用于复杂样品中双歧杆菌属定量。     

Fetal Genotyping in Maternal Blood by Digital PCR: Towards NIPD of Monogenic Disorders Independently of Parental Origin

PurposeTo date, non-invasive prenatal diagnosis (NIPD) of monogenic disorders has been limited to cases with a paternal origin. This work shows a validation study of the Droplet Digital PCR (ddPCR) technology for analysis of both paternally and maternally inherited fetal alleles. For the purpose, single nucleotide polymorphisms (SNPs) were studied with the only intention to mimic monogenic disorders.MethodsNIPD SNP genotyping was performed by ddPCR in 55 maternal plasma samples. In 19 out of 55 cases, inheritance of the paternal allele was determined by presence/absence criteria. In the remaining 36, determination of the maternally inherited fetal allele was performed by relative mutation dosage (RMD) analysis.ResultsddPCR exhibited 100% accuracy for detection of paternal alleles. For diagnosis of fetal alleles with maternal origin by RMD analysis, the technology showed an accuracy of 96%. Twenty-nine out of 36 were correctly diagnosed. There was one FP and six maternal plasma samples that could not be diagnosed.DiscussionIn this study, ddPCR has shown to be capable to detect both paternal and maternal fetal alleles in maternal plasma. This represents a step forward towards the introduction of NIPD for all pregnancies independently of the parental origin of the disease.     

A strategy for single nucleotide polymorphism analysis of chimerism for somatic cell therapy

Background aimsChimerism is an important outcome measure in hematopoietic cell transplantation as well as somatic cell therapy. Commonly used methods to estimate chimerism are restricted by either gender or inefficient sensitivity. In principle, real-time polymerase chain reaction (PCR)-based assays can be used to assess single nucleotide polymorphisms (SNP), which are a vast resource of molecular markers, and such assays demonstrate a substantially higher sensitivity (0.001%), but the specificity is unclear because of a low-level signal from mismatched sequences.MethodsIn this study, we cloned 14 pairs of SNP selected from the SNP HapMap database and examined the specificity and sensitivity of their detection by real-time PCR using two primer/fluorescent probe pairs to allow genotyping of the two possible variant alleles. Clinical donor–recipient pairs from 18 families were used to explore the efficacy of using SNP assays to measure chimerism.ResultsWe found that the polymorphic nucleotide influences the ability to distinguish the signal generated by the target and mismatched sequences. Moreover, the specific fluorescent reporter probe can affect the difference in signal intensity between the target and mismatched sequences. Real-time PCR SNP assays can attain a sensitivity of 0.1–0.5% with 100% specificity. When comparing possible clinical donor–recipient pairs, we found an average 3.3 out of 14 SNP were informative.ConclusionsBy optimal selection of the polymorphic sequences and fluorescent reporter, the real-time PCR SNP assay is superior to the short-tandem repeat chimerism assay and broadly applicable. This strategy may be applied in future clinical trials of bone marrow cell therapy.     

High-throughput genotyping system as a robust and useful tool in oncology: Experience from a single institution

Background and aimSingle nucleotide polymorphisms (SNPs) are substitutions of one base for another in the gene sequence and conforms the basis for pharmacogenetics and the development of personalized medicine. Many methods have been developed for SNP genotyping. The aim of the present study was to validate the use of a novel high-throughput genotyping system.MethodsFive SNPs (rs25487, rs25489, rs1799782, rs13181, and rs11615) were genotyped in 118 cancer patients using the classical method PCR restriction fragment length polymorphism (RFLP) and the high-throughput, automated assay Biotrove OpenArray^® NT Cycler, trying to explore the feasibility and reproducibility of the OpenArray system in the context of oncology.ResultsThe call rates obtained ranged from 95.7 to 100% for both techniques. The percentage of overlapping ranged from 96.2 to 100% among both assays, showing a high reproducibility between the techniques.ConclusionThese findings, together with the low-cost and the simple and fast work flow, suggest that the OpenArray system is a robust and easy methodology for genotyping in the field of oncology.     

Comparison of the SuperARMS and Droplet Digital PCR for Detecting EGFR Mutation in ctDNA From NSCLC Patients

BACKGROUND: Liquid biopsy is emerging as an important approach for tumor genotyping in non-small cell lung cancer, ddPCR and SuperARMS are both methods with high sensitivity and specificity for detecting EGFR mutation in plasma. We aimed to compare ddPCR and SuperARMS to detect plasma EGFR status in a cohort of advanced NSCLC patients. METHOD: A total of 79 tumor tissues and paired plasma samples were collected. The EGFR mutation status in tissue was tested by ADx-ARMS, matched plasma was detected by ddPCR and SuperARMS, respectively. RESULTS: The EGFR mutation rates were identified as 64.6% (tissue, ARMS), 55.7% (plasma, ddPCR), and 49.4% (plasma, Super ARMS), respectively. The sensitivity of ddPCR was similar with Super-ARMS in plasma EGFR detection (80.4% vs 76.5%), as well as the specificity (89.3% vs 100%). And the McNemar’s test showed there was no significant difference (P = .125). The concordance rate between SuperARMS and ddPCR was 91.1%. A significant interaction was observed between cfDNA EGFR mutation status and EGFR-TKIs treatment tested by both methods. CONCLUSION: Super-ARMS and ddPCR share the similar accuracy for EGFR mutation detection in plasma biopsy; both methods predicted well the efficacy of EGFR-TKIs by detecting plasma EGFR status.     

A novel genotyping method for detection of the CRHR1 (rs1396862: C>T) gene variation among North Indian population

Today, the genomic revolution in epidemiology, medicine and population based genetic association studies are results of on-going refocusing efforts toward development of inexpensive and accurate techniques for SNP genotyping. Despite this considerable gain, high throughput and routinely applicable newer SNP detection techniques are still needed. Therefore, aim of this study was to develop and validate a simple, rapid and inexpensive restriction enzyme based method for genotyping of corticotrophin-releasing hormone receptor1 (CRHR1; rs1396862: C>T) gene variant. This polymorphism has been investigated in a variety of psychiatric and association studies of asthma. A total of 250 healthy volunteers were recruited from same ethnicity and their blood DNA samples were employed for genotyping. Primers were designed using Batch primer3 Software. Specificity and functionality of primers were tested with BLAST database and UCSC In-silco PCR respectively. The lake of a PstI recognition site was seen with T allele. The allele frequencies for rs1396862: C>T were 0.88 (C allele) and 0.12 (T allele). We get 100 % concordant genotyping results for sequencing and PCR–RFLP. This newer genotyping approach lowers the cost and increased the speed. It is particularly useful for small basic research studies of complex genetic disorder.     

A single multiplex PCR and SNaPshot minisequencing reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

SNaPshot minisequencing reaction is in increasing use because of its fast detection of many polymorphisms in a single assay. In this work we described a highly sensitive single nucleotide polymorphisms (SNPs) typing method with detection of 42 mitochondrial DNA (mtDNA) SNPs in a single PCR and SNaPshot multiplex reaction, in order to allow haplogroup classification in Latin American admixture population. We validated the panel typing 160 Brazilian individuals. Complete SNP profiles were obtained from 10 pg of total DNA. We conclude that it is possible to build and genotype more than forty mtDNA SNPs in a single multiplex PCR and SNaPshot reaction, with sensitivity and reliability, resolving haplogroup classification in admixture populations.     

SYBR green dye-based probe-free SNP genotyping: Introduction of T-Plex real-time PCR assay

Single-nucleotide polymorphism (SNP) genotyping is widely used in genetic association studies to characterize genetic factors underlying inherited traits. Despite many recent advances in high-throughput SNP genotyping, inexpensive and flexible methods with reasonable throughput levels are still needed. Real-time PCR methods for discovering and genotyping SNPs are becoming increasingly important in various fields of biology. In this study, we introduce a new, single-tube strategy that combines the tetra-primer ARMS PCR assay, SYBR Green I-based real-time PCR, and melting-point analysis with primer design strategies to detect the SNP of interest. This assay, T-Plex real-time PCR, is based on the T_m discrimination of the amplified allele-specific amplicons in a single tube. The specificity, sensitivity, and robustness of the assay were evaluated for common mutations in the FV, PII, MTHFR, and FGFR3 genes. We believe that T-Plex real-time PCR would be a useful alternative for either individual genotyping requests or large epidemiological studies.     

Development and assessment of simple PCR markers for SNP genotyping in barley

Simple molecular marker assays underpin routine plant breeding and research activities in many laboratories worldwide. With the rapid growth of single nucleotide polymorphism (SNP) resources for many important crop plants, the availability of routine, low-tech marker assays for genotyping SNPs is of increased importance. In this study, we demonstrate that temperature-switch PCR (TSP) supports the rapid development of robust, allele-specific PCR markers for codominant SNP genotyping on agarose gel. A total of 87 TSP markers for assessing gene diversity in barley were developed and used to investigate the efficacy for marker development, assay reliably and genotyping accuracy. The TSP markers described provide good coverage of the barley genome, are simple to use, easy to interpret and score, and are amenable to assay automation. They provide a resource of informative SNP markers for assessing genetic relationships among individuals, populations and gene pools of cultivated barley (Hordeum vulgare L.) and its wild relative H. spontaneum K. Koch. TSP markers provide opportunities to use available SNP resources for marker-assisted breeding and plant genetic research, and to generate information that can be integrated with SNP data from different sources and studies. TSP markers are expected to provide similar advantages for any animal or plant species. M. J. Hayden and T. Tabone contributed equally to this work.     

Development of a Lateral Flow Strip-Based Recombinase Polymerase Amplification Assay for the Detection of Haemonchus contortus in Goat Feces

Haemonchosis remains a significant problem in small ruminants. In this study, the assay of recombinase polymerase amplification (RPA) combined with the lateral flow strip (LFS-RPA) was established for the rapid detection of Haemonchus contortus in goat feces. The assay used primers and a probe targeting a specific sequence in the ITS-2 gene. We compared the performance of the LFS-RPA assay to a PCR assay. The LFS-RPA had a detection limit of 10 fg DNA, which was 10 times less compared to the lowest detection limit obtained by PCR. Out of 24 goat fecal samples, LFS-RPA assay detected H. contortus DNA with 95.8% sensitivity, compared to PCR, 79.1% sensitivity. LFS-RPA assay did not detect DNA from other related helminth species and demonstrated an adequate tolerance to inhibitors present in the goat feces. Taken together, our results suggest that LFS-RPA assay had a high diagnostic accuracy for the rapid detection of H. contortus and merits further evaluation.     

Culture-Independent Detection and Genotyping of Mycoplasma pneumoniae in Clinical Specimens from Beijing,China

A duplex real-time PCR assay was designed for simultaneous detection and genotyping of Mycoplasma pneumoniae (M. pneumoniae). The detection/typing performance of this duplex PCR method, targeting specific genes for M. pneumoniae type 1 (mpn 459) and type 2 (mpna 5864), was compared to that of the previously published MpP1 real-time PCR assay and the genotyping method for the adhesin P1 gene (mpn 141). A total of 1,344 throat swab specimens collected from patients in Beijing, China were tested for M. pneumoniae by bacterial culture, MpP1 real-time PCR assay, and our duplex PCR assay, and positive detection rates of 26.9%, 34.4%, and 33.7%, respectively, were obtained. The duplex PCR method demonstrated high sensitivity and accuracy for detecting and genotyping M. pneumoniae, and significant differences in genotyping ability were observed when compared to the conventional P1 gene-based method. M. pneumoniae type 1 was the predominate genotype from 2008 to 2012 in Beijing, and a shift from type 1 to type 2 began to occur in 2013. To our knowledge, this is the first reported incidence of a type shift phenomenon of M. pneumoniae clinical isolates in China. These genotyping results provide important information for understanding recent changes in epidemiological characteristics of M. pneumoniae in Beijing.