HCV基因分型检测Oligo芯片的制备

目的 研制丙型肝炎病毒（HCV）基因分型寡核苷酸（Oligo）芯片并进行杂交验证。方法 分别对BLAST检索所得的HCV4个亚型型特异序列逐一进行分析，设计长度均一的60mer Oligo探针，用芯片点样仪将设计好的探针打印到玻片上制备成基因芯片。结果 杂交结果显示，Oligo芯片检测HCV各基因亚型的效果较佳。结论 制备的长链Oligo分型芯片检测敏感性、特异性均为满意，为下一步集合更多种肝炎病毒的联合检测与分型打下基础。     

寡核苷酸芯片用于HLA-B分型的初步研究

HLA基因具有复杂的多态性。本研究根据HLA-B基因序列的多态性，设计了一套寡核苷酸探针，井用基因芯片点样仪点样到醛基修饰的载玻片上，制成寡核苷酸芯片用于HLA-B基因的分型。本研究以克隆井已测序的HLA—B基因序列作为标准样品，经PCR分别扩增其具有多态性的第二和第三外显子，PCR扩增产物与芯片进行杂交，利用分析软件将杂交模式转变成为HLA—B基因型。结果表明，利用芯片进行分型的结果与标准样品序列结果完全符合。结论：寡核苷酸芯片用于HLA—B基因分型是一种简便、快捷和有前景的方法。     

寡核苷酸芯片用于HLA-A基因分型的研究

目的 应用寡核苷酸芯片分型方法，对HLA-A基因进行分型研究。方法 采用不对称PCR方法，扩增HLA-A基因的第2，3外显子，荧光标记扩增产物，作为杂交模板。设计分型检测寡核苷酸探针，制备HLA-A基因分型检测芯片。采取差异选择法，筛选强杂交信号和高特异性的分型探针。探索了探针长度、探针位置等对杂交信号的影响。杂交结果经荧光扫描，并用分型软件分析判断阳性探针和HLA-A基因型。结果 30例临床样本芯片分型结果与PCR-SSP及DNA测序分型结果相符。结论 采用寡核苷酸芯片技术对HL-A基因分型是种好的方法，具有测速快、成本低、高通量的优点。     

革兰氏阴性菌耐药基因检测芯片的研制及应用

目的探讨应用基因芯片对革兰氏阴性菌耐药基因进行快速检测的方法,并进行鉴定。方法根据GenBank上已发表的革兰氏阴性菌耐药基因盒不同亚型的基因序列,参考各亚型cDNAs区域的保守序列设计了针对16种耐药基因的特异探针,制备了革兰氏阴性菌耐药基因分型鉴定基因芯片。从16株临床分离的菌株中提取细菌基因组DNA,随机引物标记后与芯片进行杂交,同时与DNA测序法比较。结果用于评价革兰氏阴性菌耐药基因芯片的阳性菌株均出现良好的特异性杂交信号。结论该基因芯片可实现对革兰氏阴性菌耐药基因的快速检测。     

人白细胞抗原B位点基因芯片分型技术研究

目的 探讨基因芯片技术在进行北方汉族人群人白细胞抗原B位点（HLA-B）分辨度分型的价值.方法 根据中国北方汉族人群HLA-B常见基因位点及临床分型分辨度特征,设计特异性寡核苷酸中分辨度分型探针,制成HLA-B基因分型芯片.采用荧光标记引物和不对称聚合酶链反应（PCR）扩增HLA-B 2、3外显子,产物与芯片探针杂交后经荧光扫描,并用特定软件分析判断阳性探针,以确定样品基因型.结果 用中分辨度探针从30份北方汉族人标本中可分出HLA-B 7～83范围的42个B抗原等位基因,与顺序特异引物聚合酶链反应（PCR-SSP）分型方法对比,多检出3个HLA-B14、73和82新等位基因.结论 HLA-B基因芯片具有较高的精确度和特异性,可一张芯片多人份检测,适合用于临床HLA-B抗原分型.     

基因芯片在人白细胞抗原A19组基因快速分型中的应用研究

目的　建立基因芯片快速检测人白细胞抗原A1 9(HLA A1 9)分型方法。方法　利用基因芯片技术 ,根据HLA A位点不同基因亚型的独特序列设计探针 ,制成分型芯片 ;待检测样品经聚合酶链反应 (PCR)标记上荧光之后 ,与芯片进行杂交 ,根据杂交产生的荧光信号值 ,分析确定样品A位点的基因亚型。对 1 2 0份移植供受者的HLA A基因分型 ,并对 1 1份样品作基因测序。结果　仅用 2 5h ,HLA A1 9基因分型芯片可准确分辨出A1 9抗原 9大类 (A2 90 1、A2 9XX、A30 0 1、A30 0 6、A30XX、A31、A32、A34和A74 )。结论　HLA A1 9组基因芯片分型方法 ,分辨率高、特异性强、重复性好、操作简便、结果直观 ,适于临床应用     

丙型肝炎病毒1b亚型诊断芯片的制备与实验室研究

目的建立一种简单有效的制备、筛选丙型肝炎病毒(HCV)1b亚型cDNA基因芯片探针的技术。方法应用cDNA文库法制备芯片探针，限制性内切酶Sau3AI消化HCV-1b全长cDNA。所得的酶切片段在72℃补平加单个碱基A，然后与pMD18-T载体连接，AT克隆，用载体引物进行PCR初步鉴定，并测序。将筛选出的片段打印在氨基修饰的玻片上制备成cDNA芯片并进行杂交验证分析。样品标记采用限制性显示PCR(Restriction Display PCR RD—PCR)技术。结果应用cDNA文库法，共得到22大小相对一致(250—750bp)的基因片段，序列分析表明，均属于HCV—1b基因，可以作为诊断芯片探针；芯片杂交结果显示，样品和诊断基因芯片杂交的敏感性和特异性均佳。结论用cDNA文库法收集片段是一种快速、简便制备芯片探针的实用方法；制备的诊断芯片可以用于检测HCV-1b RNA，具有敏感、检测结果较为可靠的优点。     

基因芯片法和线性探针法在HCV基因分型的应用比较

目的 评价基因芯片技术在丙型肝炎病毒(HCV)基因分型中的临床应用价值。方法 对HCVRNA阳性血清用基因芯片法和线性探针法进行基因分型。结果 基因芯片法和线性探针法的符合率是100％。结论 基因芯片法的准确率与国外第二代线性探针法试剂盒相同，而操作简单，特别是能准确地检出HCV混合感染，具有良好的应用前景。     

寡核苷酸DNA芯片用于ABO血型基因分型的研究

目的 对寡核苷酸芯片用于ABO血型基因分型的技术进行研究。方法 常规的酚／氯仿法提取标准血样基因组DNA，在ABO座位的外显子6和7区域各设计一对引物，经PCR扩增基因组相应区段并用Cy5-dCTP进行标记。设计寡核苷酸分型探针，将探针固定在APS－PDC法制作的DNA芯片上，用标记的PCR产物与之杂交，扫描仪对杂交结果进行扫描，Imagene软件对杂交图象进行分析。结果 本实验共检测了100份已知血型血样的ABO基因型，结果证明本实验研制的ABO基因分型芯片快速、准确、灵敏。结论 寡核苷酸DNA芯片用于ABO的基因分型与其他方法相比更具有灵敏、直观、高通量和集成化的优势。     

乙型脑炎病毒寡核苷酸基因芯片研究

目的:制备检测乙型脑炎病毒寡核苷酸(oligo)基因芯片。方法:应用生物信息学软件设计60-meroligo探针用于制备基因芯片,乙型脑炎病毒(JEV)基因片段经限制性显示技术扩增标记,用于芯片杂交,清洗和干燥后对芯片进行扫描和数据分析。结果:大部分oligo探针都能特异性与相应样品杂交,呈现阳性荧光信号,而阴性对照和空白对照则基本不能检测到荧光信号。结论:实验中建立的oligo基因芯片检测病原体方法可行,具有应用于临床诊断的前景。     

16S rDNA基因芯片检测临床常见感染性细菌

目的 提高细菌和临床微生物检测的速度和准确性，建立含有20种细菌探针在内的感染性细菌检测用基因芯片模型。方法 使用16S rDNA克隆探针和合成的寡核苷酸探针两种，利用点样仪制成基因芯片。细菌DNA经过16S rDNA通用引物扩增后与芯片上的探针杂交，然后用荧光扫描仪检测信号。结果 基因芯片能够用于细菌检测，克服探针具有广泛和灵敏的特点，但是交叉反应明显；寡核苷酸探针具有较高的特异性，但是灵敏度稍差。结论 cDNA探针和寡核苷酸探针结合或设计几个不同的探针来指向同一株细菌很可能是将来基因芯片的检测方向。     

肽核酸芯片技术初探

目的 研究解决以DNA为探针的寡核苷酸芯片特异性、重复性差等问题。方法 用肽核酸（PNA）探针制备PNA芯片，以荧光标记的寡核苷酸（ODN）及HBV DNA的PCR产物为检测对象，探讨PNA芯片的杂交条件。用PNA芯片检测HBV DNA及单碱基突变，并与DNA芯片做一对比。结果 PNA探针对单碱基突变的识别力比相应的DNA探针更强；用其检测HBV DNA，得到了与常规PCR试剂盒一致的检测结果。结论 PNA探针独特的结构决定了它在杂交中具有与靶DNA结合的稳定性高及特异性强等优点。     

菌落原位杂交法检测革兰阴性菌Ⅰ类整合子的研究

目的用菌落原位杂交法检测革兰阴性菌中Ⅰ类整合子。方法制备Ⅰ型整合酶基因的特异性探针,采用菌落原位杂交法检测革兰阴性菌中的Ⅰ型整合酶基因,并与PCR的检测结果比较。结果40株多重耐药的革兰阴性菌中,经菌落原位杂交法检测出有18株含Ⅰ类整合子,另外22株杂交结果阴性,与PCR法检测结果一致。结论菌落原位杂交法可替代PCR法检测革兰阴性菌Ⅰ型整合酶基因。     

Blocking oligo--a novel approach for improving chip-based DNA hybridization efficiency

For most of the commonly used DNA chips, the probes are usually single-stranded oligonucleotides and the targets are double-stranded DNAs (dsDNAs). Only one strand of the DNA serves as the target while the other competes with the probes immobilized on the chip for the target and therefore is regarded as the interfering strand. In this report, a novel technique was developed for improving the hybridization efficiency on DNA chips by using blocking oligos, which is complimentary to the target interfering strand to reduce the influence of the interfering strand. The hybridization efficiency of dsDNA was much lower than that of single-stranded DNA (ssDNA) when synthesized DNA targets were tested on the DNA chip. Blocking oligos can improve the hybridization efficiency of dsDNA to about 2/3 that of ssDNA. Blocking oligos have also been applied to PCR products of different lengths for hybridization. The hybridization efficiency with blocking oligos is about three times higher than that without blocking oligos. We have tested PCR products of 1054 and 435 bp using our blocking procedure, and the results are consistent.     

Identification of a novel mutation in the dihydropyrimidine dehydrogenase gene in a patient with a lethal outcome following 5-fluorouracil administration and the determination of its frequency in a population of 500 patients with colorectal carcinoma

OBJECTIVES: Life-threatening toxic side-effects following 5-FU exposure have been related to deficiency of dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in its catabolism. We presently report a new DPYD gene SNP in a Spanish woman who died from multivisceral 5-FU-induced toxicity. DESIGN AND METHODS: We looked for 22 known SNPs by Pyrosequecing. Then, we sequenced the whole 23 exons of DPYD, along with adjacent intronic sequences. PCR was carried out to determine whether or not exons were deleted in the DPYD. To determine whether the predicted stop codon indeed resulted in a truncated protein, a bacterial expression vector was employed to generate the predicted protein. 500 patients were genotyped to determine allele frequency. RESULTS: A novel mutation 464 T>A was identified in DPYD gene exon 5, resulting in the replacement of leucine 155 by a stop codon in the protein. We confirmed this mutation by Pyrosequencing and its involvement by a protein truncation test. We genotyped the patient's family and the allele frequency was 0.2%. CONCLUSION: The involvement of this variant in 5-FU life-threatening toxicity supports its inclusion in pretherapeutic genetic screening.     

靶向mecA基因纳米胶体金探针快速检测耐甲氧西林金黄色葡萄球菌方法的建立

目的 用以纳米胶体金颗粒为载体的DNA探针杂交技术,建立快速、简便和特异的检测耐甲氧西林金黄色葡萄球菌(MRSA)的方法.方法 以直径60 nm的胶体金纳米颗粒与巯基修饰的2条mecA基因特异性寡核苷酸探针共价结合,制备成纳米胶体金探针.直接提取金黄色葡萄球菌的基因组DNA,经超声破碎后与纳米胶体金探针进行液相杂交.杂交产物经高速离心后再用反向色谱反应盘检测有无胶体金颗粒沉淀产生.用该法和PCR扩增试验同时检测临床分离株的mecA基因,以PCR法为金标准,评价纳米胶体金探针液相杂交法的准确性.结果 在95株临床试验菌株中PCR法共检出71株MRSA,24株MSSA.在71株MRSA中,纳米胶体金探针杂交试验有69株为阳性,敏感度为97.2%,最低检测限为4.72 fmol/L.而在24株甲氧西林敏感的金黄色葡萄球菌(MSSA)中,纳米胶体金探针杂交试验无一株阳性,特异度为100%.95株临床试验菌株中纳米胶体金探针杂交试验正确检出93株,准确度97.9%.结论 纳米胶体金探针杂交试验与PCR检测MRSA具有很好的一致性.应用胶体金探针杂交技术检测MRSA具有快速、简便、准确的特点,有望成为MRSA的快速诊断的新方法.     

肝炎基因诊断芯片同时检测慢性乙型肝炎患者血清及肝组织HBV DNA的临床研究

目的　用肝炎基因诊断芯片同时检测乙型肝炎患者血清及肝组织中HBVDNA ,并与雅培试剂、免疫组织化学和原位分子杂交法比较。方法　用点样仪将PCR扩增的HBCDNA探针点到特殊处理过的玻片上制成基因芯片 ,对 15例慢性乙型肝炎患者的血清及肝活检组织 ,分别用基因芯片、原位分子杂交法、免疫组织化学法、雅培试剂检测HBVDNA、HBcAg、HBsAg、HBeAg .结果　 15例HBsAg、HBeAg阳性的乙型肝炎血清 ,基因芯片检测均阳性。 15例肝组织标本 ,免疫组化法阳性 15例 ,原位分子杂交阳性 14例 ,基因芯片检测阳性 14例。结论　肝炎基因诊断可同时检测乙型肝炎患者血清及肝组织中的HBVDNA。     

Analysis of the DPYD gene implicated in 5-fluorouracil catabolism in Chinese cancer patients

Background and objective: 5‐fluorouracil (5‐FU) is still a widely used anticancer drug. More than 85% of the 5‐FU administered is catabolized by dihydropyrimidine dehydrogenase (DPD) in the liver. However, mutations in the DPD gene have been found to be associated with low DPD activity causing severe complications. The purpose of this study was to determine the mutation frequency of four exons in Chinese cancer patients and the relationship between genotype and DPD activity. Methods: Samples from 142 cancer patients were investigated in this study. The DPD activity was determined by reversed‐phase HPLC. Exons 2, 13, 14 and 18 were amplified by polymerase chain reaction (PCR), sequenced and analysed from both sense and antisense directions. Nonparametric one‐sample Kolmogorov–Smirnov test was used for distribution analysis; two independent samples t‐test and one‐way anova was performed for two groups and three groups analyses, respectively. Results and discussion: Plasma‐DPD activities in the 142 cancer patients followed a Gaussian distribution. The mean plasma‐DPD activity in women was lower than that in men (P = 0·006). Four mutations, 85T>C(DPYD*9A), 1627A>G(DPYD*5), 1896T>C and 2194G>A(DPYD*6), were found in the 142 cancer patients. The following mutations reported by others were not detected: 61C>T, 62G>A, 74A>G, 1601G>A(DPYD*4), 1679T>G(DPYD*13), 1714C>G, 1897delC(DPYD*3) and IVS 14 + 1G>A. No significant correlation was found between three mutations [85T>C(DPYD*9A), 1627A>G (DPYD*5) and 1896T>C], and DPD activity was found. Conclusion: No clear correlation between the mutations studied and DPD activity could be established in this study. However, larger‐scale prospective studies are needed to better assess the reported genotype–phenotype correlations.     

Surface plasmon resonance and biosensor technology for real-time molecular diagnosis of beta o 39 thalassemia mutation.

BACKGROUND: Biospecific interaction analysis (BIA) employing surface plasmon resonance (SPR) and biosensor technologies is of interest in clinical genetics. However, few data are available on its use in hereditary diseases caused by genetic mutations. AIM: The primary aim of this study was the refinement of BIA technology for use in identifying the beta o 39 mutation of the beta-globin gene, a mutation which causes a common type of beta o thalassemia. METHODS: Target-biotinylated PCR products were immobilized on streptavidin-coated sensor chips and diagnosed using SPR-based BIA performed by injecting specific oligonucleotide probes into the sensor chip. RESULTS: We demonstrated that the beta o 39 mutation can be easily and reproducibly identified during the association phase. CONCLUSIONS: This should be considered a pilot study demonstrating the ability of SPR-based BIA to detect point mutations in the beta-globin gene by real-time monitoring of hybridization between oligonucleotide probes and target-biotinylated PCR products generated from genomic DNA from normal, heterozygous individuals and homozygous beta o thalassemia patients.