Expression of O⁶-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue

The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.

利用改良Nest-tetra-primer specific PCR技术对两种石斑鱼的鉴别

云纹石斑鱼(Epinephelus moara)和褐石斑鱼(E.bruneus)为石斑鱼属内近缘种,外形相似常被混淆.本研究改良建立了Nest-tetra-primer specific PCR方法,获得了云纹石斑鱼和褐石斑鱼线粒体DNAND2基因内的3个特异性条带,分别为内参序列NC1(394bp)、特异性条带ND2-M(268bp)和ND2-B(122bp),以及核基因组中核糖体DNAITS1区的5个特异性条带,分别为内参序列NC2(588bp)、NC3(563bp),特异性条带rDNA-M(426bp)、ITS1-M(488bp)和ITS1-B(304bp).研究结果不仅为两种石斑鱼的鉴别提供了稳定、可靠、快捷的特异性分子标记,而且也为鱼类近缘种的DNA鉴别提供了新的途径.

高致病性猪繁殖与呼吸综合征病毒strand-specific荧光定量RT-PCR检测方法的建立

为了解高致病性猪繁殖与呼吸综合征病毒(PRRSV)的复制和转录机制,准确定量其在复制过程中基因组不同类型RNA(v RNA和c RNA)的含量,本研究根据PRRSV基因组Nsp12基因设计含有链特异性标签的特异性引物,建立了一种针对病毒基因组不同链RNA的strand-specific荧光定量RT-PCR方法。结果表明,以重组质粒标准品构建的标准曲线在101拷贝/μL^107拷贝/μL范围内具有良好的线性关系,对v RNA和c RNA的检测下限为7.40拷贝/μL和6.52拷贝/μL,能够特异性的区分病毒复制过程中产生的v RNA链和c RNA链,具有很高的特异性、灵敏性和重复性。HP-PRRSV感染MARC-145细胞后不同时间段检测v RNA和c RNA含量显示,病毒不同链RNA含量在感染过程中持续增加,v RNA和c RNA含量在感染后24 h约为7.0×107拷贝/μL和7.2×106拷贝/μL。本研究建立的方法可以用于区分和定量PRRSV在复制过程中产生的不同链RNA,为研究病毒的复制和致病机理提供了一种有效的检测手段。

基于新型高保真Taq酶的多重等位基因特异性PCR方法的建立与应用

在8号染色体存在部分替换片段的小鼠上挑选3个SNP位点进行初步方案检验,包括高保真Taq酶的特异性测试以及引物浓度对分型结果的影响,并评价该方法的灵敏度;之后应用该技术检测野生1号染色体替换系小鼠上的9个SNP的不同样本。对Taq酶的特异性研究表明:引物在未引入突变的前提下检测结果与测序结果保持一致;引物稀释实验说明浓度最低为0.003~0.006μmol/L,灵敏度的检测结果表示最低浓度检测限可达0.07 ng/μL以下。最后检测得到5种标准样本的基因型,其代表9个SNP的组合。成功验证并建立该新型高特异性Taq酶应用于多重等位基因特异性PCR实验的总体流程。提供一套具备高特异性、能够针对多个SNP位点进行检测的低成本方案,对多重等位基因特异性PCR技术的开发同样具有参考价值。

荧光定量PCR法鉴别蛤蚧定喘丸中醋鳖甲

目的建立蛤蚧定喘丸中醋鳖甲的DNA分子鉴定方法。方法根据鳖、佛罗里达鳖线粒体基因序列的差异设计特异性引物TS和AF,进行荧光定量PCR和方法学考察,然后对市售蛤蚧定喘丸中醋鳖甲进行鉴定。结果含有鳖、佛罗里达鳖的DNA样品仅与相应引物扩增后可检测到荧光信号,有特定温度单一峰,T_(m)分别为77.45、79.72℃。该方法的检测限为10 copies/μL,重复性较高(CV<1%)。10批蛤蚧定喘丸中有9批仅检出鳖DNA,被鉴定为正品;1批同时检出鳖DNA和佛罗里达鳖DNA,被鉴定为掺伪品。结论本研究所建立的方法可快速、可靠地鉴定蛤蚧定喘丸中醋鳖甲炮制品成分,有助于监管含醋鳖甲成方制剂的质量,并为成方制剂中动物药成分的专属性鉴定研究提供新途径。

应用Q-PCR定性检测KIR基因有无方法的建立

目的建立定性检测KIR基因有无的Q-PCR方法。方法根据高分辨水平中国人群KIR等位基因的多态性,并参考国际IPD-KIR数据库,针对16种KIR基因及2DS4-Normal、2DS4-Deleted两种亚型,设计KIR基因特异性引物用于Q-PCR扩增反应;同时设置一孔阴性对照、一孔阳性对照(特异性扩增人体生长激素HGH基因片段),以监控假阳性、假阴性的结果。为验证Q-PCR方法的可靠性,随机选择302份已采用KIR PCR-SSP商品化试剂盒检测的标本,采用Q-PCR方法盲检和对比。结果300人份的Q-PCR检测结果与已知的PCR-SSP检测结果相符,有2份标本结果不一致,其中1例标本的2DS5基因Q-PCR检测结果为阴性,而PCR-SSP检测结果为阳性;另一例标本2DS1基因Q-PCR检测结果为阳性,而PCR-SSP检测结果为阴性。对2份标本分别进行2DS5、2DS1基因测序分型,证实Q-PCR定性检测结果正确。结论本文建立的KIR Q-PCR方法结果准确、可靠,可用于KIR基因有无的定性检测。

Use of species-specific PCR for the identification of 10 sea cucumber species

We developed a species-specific PCR method to identify species among dehydrated products of 10 sea cucumber species.Ten reverse species-specific primers designed from the 16 S rRNA gene,in combination with one forward universal primer,generated PCR fragments of ca.270 bp length for each species.The specificity of the PCR assay was tested with DNA of samples of 21 sea cucumber species.Amplification was observed in specific species only.The species-specific PCR method we developed was successfully applied to authenticate species of commercial products of dehydrated sea cucumber,and was proven to be a useful,rapid,and low-cost technique to identify the origin of the sea cucumber product.

Species-specific PCR-based assays for identification and detection of Botryosphaeriaceae species causing stem blight on blueberry in China

Botryosphaeriaceae species are important causal agents of blueberry stem blight worldwide. Blueberry stem blight has become an important disease, potentially affecting the quality and production of blueberries in China. It is difficult and time-consuming to identify at the species level using morphological methods. The aim of this study was to develop polymerase chain reaction（PCR） assays for the diagnosis and early detection of latent infections of blueberry stems by Botryosphaeria spp. Species-specific primers, based on the ribosomal DNA internal transcribed spacer region and β-tubulin gene, were designed and selected for use in PCR assays. Three primer pairs, Lt347-F/R for Lasiodiplodia theobromae, Np304-F/R for Neofusicoccum parvum and FaF/Bt2b for Botryosphaeria dothidea, successfully amplified specific PCR fragments of different sizes on pure cultures or from blueberry stems inoculated and naturally infected blueberry plants with three pathogens, respectively. These primers did not amplify any PCR fragments from other blueberry stem disease-associated pathogens, such as Phomopsis spp. and Pestalotiopsis spp. This PCR protocol could detect as low as 1 00 pg to 1 ng of purified fungal DNA. This PCR-based protocol could be used for the diagnosis and detection of these pathogens from pure cultures or from infected blueberry plants.

Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer(ITS)region of peritrich ciliates in environmental samples

Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifi cations of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specifi c PCR primers were newly designed to amplify a fragment including the internal transcribed spacer(ITS) region of ribosomal rDNA from environmental samples. The primers showed high specifi city in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28 S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18 S rDNA in peritrichs. The newly designed specifi c primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in dif ferent systems.

大肠杆菌耐药基因TD-PCR检测方法的建立及应用

为建立一种检测大肠杆菌主要耐药基因(ARG)的通用降落PCR(TD-PCR)方法,本研究参考大肠杆菌标准菌株K12 MG1655 yeeJ基因序列设计合成不同Tm值引物、不同产物长度引物、不同长度引物,并参考Gen Bank中登录的β-内酰胺酶、氨基糖苷类、酰胺醇类、喹诺酮类和黏菌素类部分ARG序列设计合成共29对引物。以大肠杆菌标准菌株K12 MG1655总DNA为模板,采用TD-PCR和普通PCR方法,分别利用不同Tm值引物(P1~P8)、不同产物长度引物(P9~P16)、不同长度的引物(P17~P22)分别扩增yeeJ基因,均用于检测TD-PCR方法的特异性;利用引物P5扩增yeeJ基因,根据目的条带平均亮度值,对TD-PCR方法的扩增产物量分析。不同Tm值引物的扩增结果显示,TD-PCR方法中利用8对不同Tm值引物扩增后均出现目的条带,且均无非特异性条带,而普通PCR方法仅有部分引物扩增后出现目的条带,表明以不同Tm值引物扩增时,TD-PCR方法特异性更强,且适用引物Tm值范围较广。不同产物长度扩增结果显示,TD-PCR方法中利用8对引物扩增后均出现相应目的条带,且无非特异条带,而普通PCR方法虽然均出现目的条带,但部分引物扩增后还出现了非特异性条带,表明以不同产物长度引物扩增时,TD-PCR方法的特异性更强。不同长度的引物扩增结果显示,TD-PCR方法中利用6对不同长度的引物扩增后均出现目的条带,且无非特异条带,而普通PCR方法在52℃退火温度时引物P22出现非特异性扩增,表明TD-PCR方特异性更强。扩增产物量分析结果显示,TD-PCR和普通PCR方法扩增的目的条带平均亮度值均随反应总循环数的增加而上升,但普通PCR方法扩增的产物目的条带平均亮度值始终高于TD-PCR,表明TD-PCR方法的扩增产物量低于普通PCR。以24株临床分离的鹅源大肠杆菌总DNA为模板,采用TD-PCR和普通PCR方法,分别对β-内酰胺酶、氨基糖苷类、酰胺醇类、喹诺酮类和黏菌素类共29种ARG进行检测。结果显示,TD-PCR方法对其中25种ARG的检测结果与普通PCR相同,二者符率为100%;而对另外4种ARG检测结果显示,与普通PCR相比,TD-PCR方法的非特异条带明显减少,且无假阳性。表明TD-PCR在检测ARG方面比普通PCR具有更强的特异性。本研究建立了一种特异性较强的检测大肠杆菌主要ARG的TD-PCR通用方法,为细菌耐药性的研究提供了便捷高效的检测技术手段。

Identification of various Biomphalaria alexandrina strains collected from five Egyptian governorates using RAPD and species-specific PCR techniques

The first generation of Biomphalaria snails collected from five Egyptian governorates (Giza, Fayoum, Kafr El-Sheikh, Ismailia and Damietta) were sub-jected to species-specific PCR assays and the results showed that snails collected from the field were B. alexandrina, and there was no evidence for the pres-ence of B. glabrata. The snails were subjected also to RAPD- PCR technique. The results showed that dif-ferent fingerprints with each B. alexandrina strain were produced with varying numbers of bands rang-ing in size from 123.6 to 796.6 bp depending on the snail strain and the primer used. Many specific bands were obtained with the four primers in each strain. Primer OPA-1 amplified the highest number of spe-cific bands (26 bands) and gave the highest poly-morphism among the primers used (100% polymor-phism). The estimated similarity coefficients among B. alexandrina strains based on the RAPD-PCR pro-files ranged from 0.56 to 0.72. The highest similarity coefficient (0.72) was recorded between the strains of Ismailia and Kafr El-Sheikh, while the lowest coeffi-cient (0.56) was reported between the strains of SPSC and Ismailia.

Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification

We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.

Identification of <i>JAK2</i>(V617F) Mutation in Myeloproliferative Neoplasms by Using Allele Specific Polymerase Chain Reaction (AS-PCR)

Myeloproliferative neoplasms (MPNs) are a group of clonal haematopoietic stem cell disorders characterized by the proliferation of one or more myeloid cell lineages. According to WHO classification, the Janus associated kinase 2 (JAK2) V617F mutation is one of the major diagnostic criteria in BCR-ABL1 negative myeloproliferative neoplasms. The aim of this study is to detect the JAK2 (V617F) mutation in patients with myeloproliferative neoplasms to get accurate diagnosis and proper management. A total of 90 clinically diagnosed MPN patients attending to Department of Clinical Haematology, Yangon General Hospital were enrolled in this study. The mean age was 53.4 ± 14 years which ranged from 16 to 81 years old and male and female ratio was 2.4:1. The identification of JAK2 (V617F) point mutation was found to be positive in 44/90 MPN patients (48.9%). According to MPN subtypes, the JAK2 mutation positivity was found in 19 out of 46 polycythemia vera patients (41.3%), 17 out of 25 essential thrombocythemia patients (68%), 8 out of 15 primary myelofibrosis patients (53.3%), 0 of 4 others myeloproliferative neoplasms (0%). Confirmation of each of nine JAK2 mutation positive and negative samples was done by Sanger sequencing. The arterial or venous thrombotic attack was found in 32/44 JAK2 mutation positive cases (72.7%) and 12/44 JAK2 mutation negative cases (27.3%). The association between thrombotic attack and presence of JAK2 mutation was statistically significance with p = 0.000. The diagnosis of myeloproliferative neoplasms mainly relies on the molecular genetics according to WHO classification. The Allele specific PCR reaction is sensitive, simple test and relatively cost-effective. Therefore, the identification of JAK2 (V617F) somatic point mutation by AS-PCR should be implemented as a routine diagnosis procedure for patients with chronic and suspected myeloproliferative neoplasms.

Method for Solving Non-specific Amplification Interference of Fluorescence Quantitative PCR in Gene Detection

Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.

转基因玉米ND207转化事件特异性定性PCR检测方法及其标准化

转基因玉米ND207是中国农业大学研发的转mCry1Ab和mCry2Ab基因的抗虫玉米,本研究的目的是建立ND207转化事件特异性定性PCR检测方法。根据研发者提供的引物进行PCR扩增,PCR产物测序分析,获得了5’端旁侧序列262 bp,包括109 bp的载体序列和153 bp的玉米基因组序列, 3’端旁侧序列316 bp,包括76 bp的载体序列和240bp的玉米基因组序列。针对两端旁侧序列设计14条引物,组成25对引物组合进行引物筛选,选中3’端一对最佳引物优化PCR反应体系及反应条件,建立ND207的转化事件特异性定性PCR检测方法, PCR产物片段大小为166 bp。经过测试,该方法的检出限为0.1%,相当于20个拷贝的ND207特异分子片段。国内8家转基因生物安全检测机构对本方法进行了特异性测试、检出限测试和再现性测试,循环验证报告显示:该方法符合国家标准方法的各项要求,可在检测行业推广应用。ND207转化事件特异性定性PCR检测方法的建立,为我国ND207及其衍生品系的安全监管提供技术支撑。

4种牛分枝杆菌特异性基因PCR检测方法的比较与评价

牛分枝杆菌是严重危害养牛业的重要人兽共患病原微生物,PCR是检测该病原的有效手段之一。本研究通过筛选最佳的PCR检测方法,以期为临床监测牛分枝杆菌感染提供技术参考。针对牛分枝杆菌4种常见的特异性基因(16S rRNA、IS6110、IS1081和Mpb64)分别设计引物,通过温度梯度PCR法确定最适退火温度;采用牛分枝杆菌参考株核酸确定PCR最小检测限,同时运用牛分枝杆菌国内参考株、牛分枝杆菌国际参考株、禽分枝杆菌、胞内分枝杆菌、副结核分枝杆菌、羊种布鲁菌以及牛种布鲁菌进行特异性比较试验;最后运用人工模拟牛分枝杆菌感染组织样本(肺脏、淋巴结、奶样)比较不同PCR方法检测效果。4对引物以60℃为退火温度时均能检测分枝杆菌,其中IS6110、IS1081和Mpb64基因引物可以特异性检测牛分枝杆菌。IS6110和IS1081基因引物对牛分枝杆菌基因组的检测灵敏度最高,可达10^(-10) ng/μL;IS6110基因引物在肺脏和淋巴结中牛分枝杆菌检测灵敏度可达10~6 CFU/mL以上,而Mpb64基因引物对奶样中牛分枝杆菌的检测灵敏度可达10~2 CFU/mL。本研究筛选出相对适宜的牛分枝杆菌PCR检测方法,为牛结核病病原学检测和临床应用提供技术支持。

大豆根瘤菌菌株5873 PCR快速检测方法的建立及应用效果评价

【目的】大豆根瘤菌(Bradyrhizobium japonicum)是微生物肥料重要的功能菌种之一,可通过生物固氮为大豆生长提供氮素,实现节本增效,菌株5873作为其典型代表,已广泛应用于农业生产。筛选并鉴定其特异引物,建立大豆根瘤菌5873菌株水平的快速检测方法,对微生物肥料生产菌种鉴定、产品质量检测和功能评价至关重要。【方法】以商业化菌株大豆根瘤菌5873为供试材料,基于其全基因组序列和NCBI数据库中大豆根瘤菌种内参比菌株相关序列,以及与其高度同源(基因组ANI值大于99.95%)的大豆根瘤菌USDA 6T差异片段,通过多重序列比对,进行特异引物设计和筛选,获得特异性引物对,并通过对PCR反应条件/体系优化、特异性及灵敏度检测,建立大豆根瘤菌5873的快速检测方法。然后,采用盆栽试验,将大豆根瘤菌5873与其他根瘤菌菌株混合接种于大豆根际,应用上述方法对大豆根瘤菌5873竞争结瘤能力进行评价和方法验证。【结果】筛选获得了一组特异引物4-4和Q1(4-4-F 5′-GATAAGGCCACGGGTGAACA-3′/4-4-R 5′-CACTCGATAAGCTCCGCTGT-3′和Q1-F 5′-CCGGTCGTGACTGGAATGAT-3′/Q1-R 5′-TCGAGGCCTACAAGAACGTC-3′),优化并建立了PCR快速检测方法,即反应体系:Premix TaqTM 12.5μL,引物各1.0μL,基因组DNA 15 ng左右,加ddH2O补足至25μL;反应条件:95℃预热5 min,94℃变性45 s,61℃退火45 s,72℃延伸60 s,30个循环,再72℃延伸10 min。通过凝胶电泳检测目的条带的有无(355和218 bp),即可实现大豆根瘤菌5873的快速检测,该方法检出灵敏度为1850 CFU/μL。此外,借助该方法可以成功地评价大豆根瘤菌5873竞争结瘤能力,与传统BOX-PCR评价结果一致。【结论】大豆根瘤菌5873快速鉴定方法的建立实现了以菌体发酵液或根瘤破碎提取液为模板,直接进行PCR扩增的鉴定操作,省去了根瘤的分离、根瘤菌分离纯化、培养、DNA提取及测序鉴定等繁琐的环节,大大减少了工作量,只需短短几个小时便可准确检测大豆根瘤菌5873,为微生物肥料中根瘤菌菌剂的产品质量检测和竞争结瘤能力评价提供了参考。

抗逆大豆IND-ØØ41Ø-5转化体特异性定量PCR检测方法的建立及其标准化

【目的】抗逆大豆IND-ØØ41Ø-5已获得中国进口加工原料安全证书,建立一种特异、准确、灵敏的抗逆大豆IND-ØØ41Ø-5转化体特异性实时荧光定量PCR(qPCR)检测方法,为抗逆大豆IND-ØØ41Ø-5在中国的安全监管提供精准测量技术。【方法】首先利用Beacon designer 8.0软件对抗逆大豆IND-ØØ41Ø-5转化体5′端旁侧序列设计18对引物探针,结合1对罗萨里奥农业生物技术学院公司提供的引物探针,随后采用qPCR技术对引物探针进行特异性筛选,遴选出1对候选引物探针;设置实时荧光定量PCR的引物/探针浓度梯度,优化qPCR方法的反应体系;设置不同类型的特异性测试样品测试方法的特异性;以纯合抗逆大豆IND-ØØ41Ø-5基因组DNA作为标准样品,梯度稀释成标准溶液模板,绘制标准曲线,考察qPCR方法的线性动态范围,配置拷贝数比值为5%、1%和0.1%的测试样品,评价定量方法的准确性;配置拷贝数比值为0.05%和0.025%的样品,测试方法的检出限;拷贝数比值为0.1%的样品经16次定量测试,确定方法的定量限;最终确定抗逆大豆IND-ØØ41Ø-5转化体特异性实时荧光定量PCR检测方法的关键技术参数。8家有资质单位对该定量PCR方法的特异性、检出限、定量限、准确性等进行验证,采用柯克伦法和格拉布斯法评估qPCR方法的重复性和再现性,利用线性最小二乘法对准确性样品测试结果的测量不确定度进行预评定。【结果】通过特异性筛查确定RBORD-F1/RBORD-R1/RBORD-P1引物探针为候选组合,建立了抗逆大豆IND-ØØ41Ø-5转化体特异性qPCR方法,扩增片段为138 bp,优化的反应体系引物终浓度为0.4μmol·L-1、探针终浓度为0.2μmol·L-1;IND-ØØ41Ø-5和Lectin标准曲线的线性动力学范围为33—83190 copies的基因组DNA,该方法可准确定量5%、1%和0.1%含量的IND-ØØ41Ø-5测试样品,偏差和相对标准偏差(RSD)均小于25%;确定检出限为0.05%、定量限为0.1%。8家实验室间联合验证该方法稳定性好、特异性强,检出限为0.05%,定量限为0.1%,且具备良好的实验室间重复性和再现性,经测量不确定度预评定后,获得5份抗逆大豆IND-ØØ41Ø-5测试样品的测量结果分别为(0.10±0.02)%、(0.53±0.09)%、(1.05±0.18)%、(2.05±0.34)%和(5.18±0.87)%,结果准确可靠。【结论】成功建立了抗逆大豆IND-ØØ41Ø-5转化体特异性实时荧光定量PCR检测方法,能够实现抗逆大豆IND-ØØ41Ø-5转化体成分的精准定量检测。

Flow-rSSO及PCR-SBT方法检测KIR基因有无的对比研究

目的研究流式磁珠序列特异性寡核苷酸探针(Flow-rSSO)杂交及测序分型(PCR-SBT)方法鉴定KIR基因有无的一致性。方法对131例汉族人群的DNA标本采用Flow-rSSO方法鉴定全部16种KIR基因的有无,并采用本实验室建立的PCR-SBT方法对14种功能性KIR基因进行高分辨水平基因检测;分析Flow-rSSO及PCR-SBT两种方法鉴定14种功能性KIR基因有无的一致性。对初检结果不一致的标本,采用同一厂家、不同批号的Flow-rSSO试剂盒进行复检,并采用序列特异性引物-PCR(PCR-SSP)方法进行检测。结果131例标本中有129例完全一致,2例不一致,占1.5%(2/131)。不一致的两例标本,1例标本3DL1基因、另1例标本2DS3及2DS5基因,其Flow-rSSO初检结果均为阴性,而PCR-SBT结果均为阳性。更换新的批号Flow-rSSO试剂盒复检,两例标本的结果均为阳性;采用PCR-SSP方法检测的结果亦为阳性。结论经Flow-rSSO试剂盒检测KIR基因有无,出现2例标本初检结果错误,提示检测KIR基因有无时,试剂的质控工作至关重要。