错配杂交化学发光法检测MTHFR基因多态性

目的建立一种基于错配杂交及化学发光技术检测亚甲基四氢叶酸还原酶（methylene tetrahydrofolate reduetase。MTHFR）基因多态性的新方法。方法针对多态位点设计两条寡核苷酸探针,分别与野生型及突变型序列互补。将2条探针分别与含多态位点C677T的PCR扩增产物杂交,根据2条探针的发光值之比确定样品的基因型。结果用本法随机检测50例DNA样品,结果3种基因型的发光值之比可以严格区分,其中野生型（C／C）15例,杂合型（C／T）28例,突变型（T／T）7例,与参考方法（PCR—RFLP）的检测结果完全一致。结论本方法操作简便、快速且费用低廉,可广泛应用于MTHFR基因突变及多态性检测。     

错配杂交化学发光法检测亚甲基四氢叶酸还原酶基因多态性

目的 建立一种基于错配杂交及化学发光技术的检测亚甲基四氢叶酸还原酶（methylenetetrahydrofolate reducatase,MTHFR）基因多态性的新方法。方法 针对多态位点设计两条寡核苷酸探针,分别与野生型及突变型序列互补。将两条探针分别与含多态位点C677T的PCR扩增产物杂交,然后用化学发光法检测,根据两条探针的发光值之比确定样品的基因型。结果 用本法随机检测了50例DNA样品,结果3种基因型的发光值之比可以严格区分,其中野生型（C／C）15例,杂合型（C／T）28例,突变型（T／T）7例,与参考方法（PCR—RFLP）的检测结果完全一致。结论 本方法操作简便、检测快速而且费用低廉,可广泛应用于MTHFR基因突变及多态性检测。     

影响亚甲基四氢叶酸还原酶和甲硫氨酸合成酶还原酶基因多态性的因素分析

目的了解影响女性亚甲基四氢叶酸还原酶(MTHFR)C677T、A1298C位点和甲硫氨酸合成酶还原酶(MTRR)A66G位点基因多态性的因素。方法采用横断面调查研究方法,以湖北省内1 902例女性为对象,采集口腔黏膜上皮细胞,提取DNA,用实时荧光定量PCR技术检测MTHFR和MTRR基因,统计分析不同民族MTHFR和MTRR基因多态性的差异,与其他地区人群的MTHFR和MTRR基因多态性进行比较,并分析不同位点基因多态性之间的关联关系。结果 (1)湖北省汉族女性和少数民族女性在MTHFR C677T和MTRR基因多态性构成差异无统计学意义(P>0.05),而MTHFR A1298C位点基因多态性构成差异有统计学意义(P<0.05)。(2)MTHFR C677T位点的纯合突变基因型TT的频率从南到北呈上升趋势,MTHFR A1298C位点的纯合突变基因型CC的频率从南到北呈下降趋势,而MTRR A66G位点基因多态性在不同地区差异不大。(3)MTHFR C677T位点的纯合突变基因型TT的频率在A1298C位点的正常型、杂合型、纯合突变型中分别为15.9%、0.1%、0.0%,差异有统计学意义(P<0.01);MTHFR C677T位点的纯合突变基因型TT的频率在MTRR A66G位点的正常型、杂合型、纯合突变型中分别为9.4%、5.9%、0.7%,差异无统计学意义(P>0.05)。结论 MTHFR A1298C位点基因多态分布存在民族差异;而MTRR C677T、A1298C位点基因多态分布存在地区差异;同一基因不同位点的基因多态性有关联。     

TaqMan-ARMS法快速检测人MTHFR基因多态性

目的利用PCR引物的错配扩增和荧光定量PCR技术,建立一种针对人亚甲基四氢叶酸还原酶(methylene tetrahydrofolate reductase,MTHFR)基因多态性的快速荧光扩增检测方法。方法收集已经测序验证的MTHFR基因C677T,A1 298C位点的野生型、杂合型和突变型样本,并据此构建野生型和突变型质粒;根据野生型MTHFR基因序列设计ARMS(扩增阻碍突变系统)引物和Taq Man探针并筛选出最合适的突变检测体系,与已知突变信息的214例样本进行比较,以验证该检测体系的可行性。结果建立的Taq Man-ARMS法性能评估优异,样本的最低检测限为10 copies/μL,样本间交叉检测无核酸扩增,体系检测阴性对照无核酸扩增;重复性及实验室内精密度结果良好,MTHFR-667位点和1 298位点重复性检测的标准差介于0.11~0.44,纯合和杂合样本的变异系数(CV)均4.52%。214例临床样本用该法检测结果与测序法的一致性为100%。结论基于Taq Man-ARMS法检测MTHFR的基因多态性方法简单,快捷,精确,适合用于临床样本快速诊断。     

新型亚甲基四氢叶酸还原酶单核苷酸多态性研究方法检测恶性血液病易感性

本研究探讨一种新型亚甲基四氢叶酸还原酶（MTHFR）单核苷酸多态性（SNP）检测方法，并用其检测恶性血液病遗传易感性。根据cDNA芯片原理制作一种目的基因芯片，利用双色荧光探针杂交进行SNP位点检测，测序法对该芯片检测结果的准确性进行验证，并以此对来自中国江苏地区的157例健康对照和127例恶性血液病患者（30例多发性骨髓瘤，28例非霍奇金淋巴瘤，22例急性淋巴细胞白血病，40例急性髓系白血病，7例慢性髓系白血病）的MTHFR基因C677T多态位点进行检测。结果表明，为野生型、杂合型和突变型的叠加荧光分别显示为绿色、黄色和红色。测序结果与芯片结果吻合。677C和677T在病例和对照组的基因频率分别为58．7％、66．9％、41、3％和33、1％，差异有显著性（χ^2=4．077，P=0．043）。677TT基因型发生MM相对风险明显增加（OR=4．21；95％CI=1．50-11．83；P=0．006）。结论：本芯片检测方法准确、高通量且价格低廉，适用于大规模样本SNP调查；C677T多态改变影响恶性血液病的发病风险。677TT基因型是MM的易感因素。     

亚甲基四氢叶酸还原酶基因多态性两种检测方法之比较

目的应用实时荧光PCR技术，建立一种快速、准确的检测亚甲基四氢叶酸还原酶（MTHFR（677C→T））基因多态性的方法。方法采用实时荧光PCR技术和聚合酶链反应-限制性片段长度多态性（PCR—RFLP）同时检测300例健康体检者MTHFR（677C→T）位点的基因多态性，并以测序佐证检测结果的准确性。结果用2种方法均检出CC、CT、TT3种基因型，基因型频率和等位基因频率2组间差异无统计学意义（基因型P〉0．05，等位基因P〉0．05）。结论实时荧光PCR法准确、快捷，适合临床对该基因位点的快速分型。     

广东地区汉族育龄妇女MTHFR基因遗传多态性

目的调查并分析广东地区汉族育龄妇女MTHFR基因多态性分布情况。方法选取2016年1月至2018年7月广东省妇幼保健院行婚检/孕检的育龄妇女13336例,采用Taqman-MGB法测定MTHFR基因C677T、A1298C位点的基因型,并采用卡方检验比较本地人群与其他人群的基因型频率/等位基因频率的分布情况。结果MTHFR基因C667T位点野生型(CC)、杂合突变型(CT)和纯合突变型(TT)分别占51.5%、38.7%和9.8%,突变T基因频率为29.2%;MTHFR基因A1298C位点中,野生型(AA)、杂合突变型(AC)和纯合突变型(CC)分别占59.3%、35.2%和5.5%,突变C基因频率为23.1%。广东地区汉族育龄妇女MTHFR基因C677T、A1298C位点基因型分布和等位基因频率分布与全国人群比较差异均有统计学意义(P<0.01)。广东地区汉族育龄妇女MTHFR基因C677T、A1298C位点的基因频率和等位基因频率与江苏、九江、齐齐哈尔和长沙等地区汉族育龄妇女人群相比差异均有统计学意义(P<0.05)。广东地区汉族育龄妇女MTHFR基因C677T、A1298C位点的等位基因频率与亚洲、东亚、南亚、欧洲、美洲地区人群等位基因频率的分布存在不同程度的差异。结论广东地区人群MTHFR基因的多态性分布与其他地区相比存在明显的差异。     

武汉地区缺血性脑卒中患者MTHFR基因多态性分布分析

目的:探讨武汉地区缺血性脑卒中患者亚甲基四氢叶酸还原酶(MTHFR)基因多态性的分布。方法:选取缺血性脑卒中患者167例为卒中组,健康体检者110例为对照组。通过基因芯片法检测缺血性脑卒中相关的MTHFR基因677位点,并按基因型别分为野生型(C677C)、杂合型(C677T)、纯合型(T677T)。结果:卒中组中野生型22.2%,杂合型43.1%,纯合型34.7%。与对照组相比,野生型显著减少,纯合突变型显著增多,有显著性差异(P0.01)。不同性别脑卒中患者在MTHFR基因分型上差异无统计学意义(P0.05)。结论:武汉地区缺血性脑卒中患者中分布有较多的纯合突变型MTHFR基因。     

SNP基因芯片方法的建立及在MTHFR多态性检测中的初步应用

目的 建立单核苷酸多态性（SNP）的基因芯片检测法,并初步应用于结直肠癌患者MTHFR基因位点C677T的多态性检测,分析其位点突变与致病性的关系.方法 采用醛基修饰玻璃基片,阵列检测亚甲基四氢叶酸还原酶（MTHFR）C677T基因型,生物素标记显色.应用该芯片检测78例结直肠癌患者及40例健康对照组MTHFR基因C677T多态性,并分析MTHFR基因多态性与结直肠癌的相关性.结果 建立检测MTHFR基因SNP的基因芯片.采用基因芯片法检测病例组中MTHFR基因的C677T位点CC、CT、TT基因型分布频率分别为38.5%、53.8%、7.7%,健康对照组中C677T位点CC、CT、TT基因型分布频率分别为35.0%、62.5%、2.5%.结论 成功建立检测SNP的基因芯片法.MTHFR基因位点C677T的基因多态性与结肠癌易感性无明显关系.     

老年动脉粥样硬化性脑梗死患者MTHFR基因多态性研究

目的探讨MTHFR基因多态性与老年动脉粥样硬化性脑梗死（ACI）的关系。方法应用PCR-RFLP检测88例ACI患者及31例对照组MTHFR基因多态性。结果ACI组与对照组MTHFR基因C677T位点各基因型频率、T等位基因频率、T／T纯合子频率差异无显著统计学差异。结论老年患者MTHFR基因C677T突变的纯合子与ACI无显著关系,不能被确定为老年ACI的独立危险因素。     

Detection of CYP I A1 polymorphisms with a colorimetric method based on mismatch hybridization

BACKGROUND: CYP I A1 polymorphisms, which have been reported to be associated with an elevated risk of lung cancer, are usually detected through conventional methods such as PCR-restriction fragment length polymorphism, allele-specific PCR and single-strand conformational polymorphism. METHODS: An assay that makes use of differences in thermal stability between perfect match and non-perfect match hybrids has been developed. Two oligonucleotides probes for each CYP1A1 polymorphic site were designed and labeled with digoxigenin. After hybridization with amplified DNA fragment, the hybrids were detected with a colorimetric method and the genotype were identified by calculating the ratio of signals obtained with two probes. RESULTS: The ratios for three genotypes obtained from 50 samples can be divided into three distinctive nonoverlapped groups when applying to m(1) and m(2) sites of CYP1A1 locus, which demonstrated the feasibility of this assay to detect CYP I A1 polymorphisms. CONCLUSION: Compared with other methods, this assay has lower cost, is fast, simple and is suitable for a screening test in routine laboratory.     

The use of real-time PCR and fluorogenic probes for rapid and accurate genotyping of newborn mice

Real-time PCR and fluorogenic probes were combined in a simple, rapid and sensitive method to genotype murine breeding stocks and their progeny for a point mutation. DNA from tail biopsies of newborn mice was mixed with amplification primers and fluorogenic hybridization probes in a PCR mixture. The primers were designed to amplify a region of the Fas-Ligand gene including the site for the gld natural point mutation. The fluorogenic hybridization probes overlaid this target sequence and were used to detect amplification of the PCR fragment as well as determine the presence of the point mutation using fluorescence resonance energy transfer (FRET). Both mutated and wild-type forms of the gene fragment were amplified as detected with real-time PCR. Melting curve profiles completed on each amplified sample revealed the genotype for each mouse. These genotypes were confirmed by sequencing the amplified fragments. These results suggest real-time spectrofluorometric PCR techniques incorporating FRET-based hybridization probes may be used for rapid, sensitive, inexpensive and reliable genotyping.     

Microtiter format for simultaneous multianalyte detection and development of a PCR-chemiluminescent enzyme immunoassay for typing human papillomavirus DNAs

BACKGROUND: To allow multianalyte binding assays, we have developed a novel polystyrene microtiter plate containing 24 main wells, each divided into 7 subwells. We explored its clinical potential by developing a PCR-chemiluminescent immunoassay (PCR-CLEIA) for simultaneous detection and typing of seven high oncogenic risk human papillomavirus (HPV) DNAs in one well. METHODS: Seven different oligonucleotide probes, each specific for a high-risk HPV genotype, were separately immobilized in the subwells. Subsequently, a digoxigenin-labeled consensus PCR amplification product was added to the main well. The PCR product hybridized to the immobilized probe corresponding to its genotype and was subsequently detected by use of a peroxidase-labeled anti-digoxigenin antibody and chemiluminescence imaging with an ultrasensitive charge-coupled device camera. Results obtained for 50 cytologic samples were compared with those obtained with a conventional colorimetric PCR-ELISA. RESULTS: The method was specific and allowed detection of 50 genome copies of HPV 16, 18, 33, and 58, and 100 genome copies of HPV 31, 35, and 45. Intra- and interassay CVs for the method were 5.6% and 7.9%, respectively. All results obtained for clinical samples were confirmed by the conventional PCR-ELISA. CONCLUSIONS: PCR-CLEIA allows rapid, single-tube simultaneous detection and typing of seven high-risk HPV DNAs with small reagent volumes. The principle appears applicable to the development of other single-tube panels of tests.     

应用DNA探针检测甲氧西林耐药葡萄球菌

目的建立一种快速检测葡萄球菌和甲氧西林耐药葡萄球菌的斑点杂交技术。方法设计金黄色葡萄球菌nuc基因、甲氧西林耐药mecA基因、葡萄球菌tuf基因的特异引物,用聚合酶链反应合成其特异DNA探针,并用生物素标记,分别与固定在硝酸纤维素膜上的标准菌株和临床分离株模板DNA杂交,观察其敏感性和特异性。结果3对引物分别扩增出270bp、310bp、370bp3种DNA探针,均具有高度特异性。50株金黄色葡萄球菌tuf、nuc基因均为阳性：mecA基因阳性者22株。30株表皮葡萄球菌tuf基因均为阳性,nuc基因均为阴性,mecA基因阳性者9株。而其他非葡萄球菌与3种DNA探针杂交结果均为阴性。该方法可检测出1 ng细菌DNA。结论斑点杂交技术检测耐甲氧西林葡萄球菌快速、有效,具有较高的应用价值。     

Real-time quantitative PCR for the detection of Streptococcus pneumoniae in the middle ear fluid of children with acute otitis media

PCR based on the amplification of pneumolysin gene fragments has previously been applied to demonstrate Streptococcus pneumoniae in clinical specimens. Here, a real-time PCR method for the detection and quantification of pneumococci by amplifying a 206-bp fragment of the pneumolysin-encoding gene is described. The amplified fragments were detected simultaneously using fluorescent-labeled sequence-specific hybridization probes. The applicability of the assay to clinical samples was evaluated by studying 50 middle ear fluid (MEF) specimens from children with acute otitis media. Twenty-six of the MEF samples were positive by real-time PCR and the numbers of genome equivalents detected varied from 90 to 88,000/microl in 17 culture-positive samples and from 1 to 1,200/microl in 9 culture-negative samples. The results were compared to culture findings and to results obtained by using agarose gel electrophoresis or Europium-labeled hybridization probes for the detection of amplification products of conventional PCR. The sensitivity and specificity of the real-time PCR assay developed in the present study compared to culture were 100 and 73%, and to conventional PCR with agarose gel and/or TRF detection 93 and 96%, respectively. The real-time PCR assay was found to be rapid, easy to use, and sensitive in detecting and quantifying pneumococci.     

Digoxygenin-labelled DNA-probe: a rapid non-radioactive method for hepatitis B virus DNA detection in serum.

The sensitivity and specificity of two non-radioactive spot hybridization assays for hepatitis B virus DNA (HBV-DNA) using biotin and digoxygenin-labelled DNA probes were investigated in parallel in 122 serum samples from patients with chronic hepatitis B and 50 controls. The results were compared with an isotopic technique using a 32P-labelled probe. HBV-DNA was detected in 56 (80%) out of 70 hepatitis B "e" antigen (HBeAg)-positive cases and in 4 (8%) out of 52 antibody to hepatitis B "e" antigen (anti-HBe)-positive cases using the digoxygenin or 32P-labelled probes. No false positives were found with either method. Using the biotin-labelled probe, 16% of sera gave discordant results, which were considered to be false positive. The time required for detection of serum HBV-DNA was 2 hours for the non-radioactive probes and 16 hours for the isotopic probes. This study suggests that the digoxygenin-labelled probe for detection of HBV-DNA is the most rapid and sensitive method for routine diagnosis of viral replication in clinical laboratories.     

白细胞介素6基因启动子区两个突变位点多态性检测方法的建立

目的建立一种快速检测IL-6基因启动子区-597G／A、-572G／C多态性的双重实时荧光PCR方法。方法采用1对引物，2对荧光标记探针，结合荧光共振能量转移原理和熔点曲线分析技术，检测IL-6基因启动子区-572G／C，-597G／A2个位点多态性。结果用建立的双重实时荧光PCR方法对123名健康查体者进行检测，发现中国汉族人IL-6基因启动子区-572位点有3种基因型，分别为GG，GC，CC型；-597位点仅发现4名为GA型，其余均为GG型，尚未发现从型。结论双重实时荧光PCR法简便快速，与其他方法比较具有准确、经济的特点，适合临床基因快速分型。     

Rapid detection of the hepatitis B virus YMDD mutant using TaqMan-minor groove binder probes

BACKGROUND: TaqMan-minor groove binder (MGB) probes were used in a real-time PCR-based assay for the rapid and accurate detection of hepatitis B virus (HBV) YMDD mutants. METHODS: TaqMan-MGB probes were designed to distinguish between wild-type (YMDD) and mutant (YVDD and YIDD) strains of HBV. The detection limit and sensitivity of the assay were determined using a dilution series of a mixture of wild-type and mutant plasmids. Serum samples collected from four patients with chronic mutant HBV infections during lamivudine therapy were analyzed using this method. RESULTS: The detection limit for YVDD and YIDD was 10 and 50 copies, respectively, whereas the sensitivity was 10% within a mixed virus population. In the clinical samples, mutant strains of HBV could be detected at levels <2.6 log copies/ml of HBV DNA. While 15 of the 21 samples tested by this method were positive for the YMDD mutant, direct sequencing and a reverse hybridization line probe assay (INNO-LiPA HBV DR v2) detected the mutant strain in only 11 and 9 samples, respectively. Moreover, the data for 6 samples analyzed by TA cloning were fully consistent with our TaqMan PCR results. CONCLUSIONS: We successfully established a sensitive and accurate assay for the YMDD mutant of HBV. This method may be useful for monitoring patients treated with lamivudine.