基于LAMP技术建立解脲支原体基因试纸条检测方法

目的 建立一种灵敏度高、特异性强、检测速度快的方法检测解脲支原体.方法 基于环介导恒温扩增技术(LAMP),根据解脲支原体序列特征设计3对引物进行解脲支原体DNA切口酶核酸恒温扩增,扩增过程在一对引物中标记生物素,随着扩增的进行生物素直接引入扩增片段中,扩增结束后产物在密闭装置中进行免疫试纸条显色反应,根据显色卡的颜色判定结果的阴阳性.结果 该技术检测解脲支原体较实时荧光PCR技术灵敏度要高10倍以上,其它病原体检测均阴性该方法特异性与实时荧光PCR技术相当.结论 恒温扩增联合试纸条技术检测解脲支原体具有较高的敏感性和特异性,检测速度快,适合各医院开展.     

真核表达载体pcDNA3.1(+)-vIL-10的构建及其在SD大鼠骨髓基质干细胞的表达

目的构建携带免疫调节因子vIL-10c DNA的真核表达载体,转染至SD大鼠骨髓基质干细胞,观察免疫调节因子vIL-10在骨髓基质干细胞的表达。方法PCR扩增原核载体PET-28α/vIL-10,获得目的基因vIL-10,目的基因在大肠杆菌中扩增后连入真核表达载体Pc DNA3.1(+),转染大鼠骨髓基质干细胞。用G418筛选得到vIL-10高表达的细胞,对照组为pcDNA3.1(+)直接转染的骨髓基质干细胞。结果经PCR、酶切、测序鉴定显示按设计构建的载体pc DNA3.1(+)-vIL-10,已成功转染骨髓基质干细胞。RT-PCR检测到510bp目的片段vIL-10在骨髓基质干细胞表达,SDS-PAGE电泳表明在骨髓基质干细胞有vIL-10蛋白的表达。结论vIL-10 mRNA和蛋白在骨髓基质干细胞中表达。     

PCR-RFLP和MLPA两种技术方法对脊髓性肌萎缩症的诊断

目的应用PCR-RFLP和多重连接依赖式探针扩增法(MLPA)技术对临床诊断为脊髓性肌萎缩症(SMA)患儿进行基因诊断,并比较两种方法检测SMN基因缺失改变的效果。方法应用DNA抽提试剂盒抽提3个SMA家系成员的外周血样品DNA,电泳检测,定量,常规PCR法扩增SMN基因的7、8号外显子,Dra I、Dde I酶切PCR产物,常规聚丙烯胺凝胶电泳法分离酶切后PCR产物,Genefinder染色检测。同时应用M LPA检测试剂盒P021进行检测。结果 PCR-RFLP显示3个家系中的患儿均纯合缺失SMN1的第7、8号外显子,MLPA检测结果与之完全相符。结论与PCR-RFLP相比,MLPA更加简便、快捷、可靠,是一种高效的遗传病基因诊断手段。     

铜绿假单胞菌外毒素衍生物PE38KDEL的原核表达载体构建及其鉴定

目的 构建铜绿假单胞菌外毒素衍生物(PE38KDEL)的原核表达载体并对其表达的蛋白进行鉴定.方法 采用PCR方法扩增本实验所需要的PE38KDEL基因片段,再通过酶切及连接反应构建原核表达载体pGEX-4T-1-PE38KDEL,重组载体经过限制性内切酶酶切、PCR扩增鉴定及DNA序列测定证实插入片段正确后,转化感受态大肠杆菌BL21,经IPTG诱导表达,表达产物经SDS-PAGE电泳后及蛋白免疫印迹法分别测定其大小和特异性.结果 经鉴定证实原核表达载体pGEX-4T-1-PE38KDEL构建成功,且在大肠杆菌BL21中获得了PE38KDEL与GST的融合表达,且表达蛋白产物的分子质量大小与预期值一致,并可被PE的特异性抗体所识别.结论 PE38KDEL在大肠杆菌中获得了高效的融合表达,为下一步研究其功能奠定了基础.     

CAD基因原核表达载体的构建及表达产物的活性鉴定

目的 构建pCool-GST-ICAD/CAD的表达载体,并在大肠杆菌BL21(DE3)中表达具有生物学活性的CAD核酸酶.方法 用PCR扩增CAD基因, 将扩增产物克隆入pCool-GST-ICAD载体中构建出pCool-GST-ICAD/CAD表达载体.经酶切和电泳鉴定正确后,在大肠杆菌BL21(DE3)中诱导表达,表达产物经亲和层析、离子交换层析及凝胶过滤等方法分离纯化,最后用SDS-PAGE和DNA降解实验进行鉴定.结果 构建了pCool-GST-ICAD/CAD原核表达载体,重组载体转化后表达出毫克级水平的GST-ICAD/CAD蛋白复合体.经分离纯化后得到纯度很好的CAD-ICAD蛋白复合体,在SDS-PAGE电泳上呈现清晰的两条蛋白带.经DNA降解实验证明纯化所得的CAD蛋白具有非特异性降解DNA的核酸酶作用.结论 成功制备了具有生物学活性的CAD核酸酶,为进一步研究细胞凋亡的作用机制提供了有效的制剂.     

恶性疟原虫FCC—1／HN株环子孢子蛋白基因分枝杆菌——大肠杆穿梭表达重组质粒的构建及序列测定

本文对恶性疟原虫环子孢子蛋白(circumsporozoiteprotein,CSP)基因片段进行克隆和序列测定。根据恶性疟原虫837株基因编码序列设计合成一对引物,采用PCR技术从恶性疟原虫FCC-1/HN株基因组DNA中特异扩增CSP基因片段的Ⅰ区、中央重复区、重复区后可变区和Ⅱ区;经纯化的扩增产物用BamHⅠ和KpnⅠ双酶切后,定向克隆入大肠杆菌——分枝杆菌穿梭表达质粒,转化感受态大肠杆菌DH5α,重组克隆经抗性筛选和快速凝胶电泳鉴定,再经PCR和酶切鉴定,并对重组子进行序列测定。结果表明从恶性疟原虫FCC-1／HN株基因组DNA中可特异扩增出约1171bp的基因片段,阳性重组质粒经双酶切和PCR鉴定与预期的结果一致,序列测定表明所克隆的基因和编码环子孢子抗原的基因片段相符。     

构建pCool-GST-ICAD/CAD并在大肠杆菌中表达CAD核酸酶及活性研究

目的：构建pCool-GST-ICAD/CAD的表达载体，并在大肠杆菌BL21(DE3)中表达具有生物学活性的CAD核酸酶。方法：用PCR扩增CAD核酸酶基因,将扩增产物克隆入pCool-GST-ICAD载体中构建出pCool-GST-ICAD/CAD表达质粒。经酶切和电泳鉴定正确后，在大肠杆菌BL21(DE3)中诱导表达，表达产物经亲和层析、离子交换层析及凝胶过滤等方法分离纯化，最后用SDS-PAGE和DNA降解实验进行鉴定。结果：构建了pCool-GST-ICAD/CAD原核表达质粒，重组载体转化后表达出毫克级水平的GST-ICAD/CAD蛋白复合体，经分离纯化后得到纯度很好的CAD/ICAD蛋白复合体，在SDS-PAGE电泳上呈现清晰的两条蛋白带，经DNA降解实验证明纯化所得的CAD蛋白具有非特异性降解DNA的核酸酶活性。结论：成功构建具有生物学活性的CAD核酸酶，有助于进一步研究细胞凋亡的作用机制。     

小鼠γ-干扰素真核表达质粒的构建和鉴定

目的克隆小鼠γ-干扰素(γ-IFN)基因,构建并鉴定小鼠γ-干扰素真核表达质粒.方法从BALB/c小鼠脾脏提取总RNA,用RT-PCR方法扩增出小鼠γ-干扰素基因, 分别用EcoRⅠ、BamHⅠ双酶切扩增片段和pcDNA3.1(-),定向克隆到真核表达质粒pcDNA3.1(-),构建成真核表达质粒pcDNA3.1(-)γ-IFN.转化产物通过PCR扩增筛选,双酶切鉴定;阳性克隆进行序列分析.结果 RT-PCR产物电泳可见一约500bp大小目的片段.PCR和双酶切电泳结果均证实已插入约500bp的γ-IFN基因片段;阳性克隆测序结果表明克隆的小鼠γ-干扰素基因完全正确.结论成功构建了小鼠γ-干扰素真核表达质粒,为进一步气道内实施哮喘小鼠基因治疗打下了坚实基础.     

载脂蛋白E基因的PCR快速分型法的建立

目的建立载脂蛋白E基因的PCR快速分型法.方法采用以PCR扩增为核心的载脂蛋白E(apoE)基因型分析方法,扩增其中含有112位和158位两个氨基酸位点之间的编码序列,产物为267bp apoE基因经扩增后,扩增产物经HhaI 内切酶消化后,然后进行5%琼脂糖凝胶电泳检测,快速鉴定apoE的基因型.结果本法检测了85名健康人apoE 基因型,求出三个等位基因E2、E3、E4的频率分别为0.0824、0.8412、0.0764.结论该方法简单、准确,适合于一般实验室开展.     

人TREM-1基因的克隆及其在大肠杆菌中的表达

目的系统地研究TREM-1的生物学功能,克隆TREM-1的cDNA,构建原核表达载体后,使其在大肠杆菌中得到表达。方法用RT-PCR从临床患者外周血白细胞中扩增TREM-1 cDNA,将其克隆到克隆载体pGEM-3Z上。经酶切及PCR扩增鉴定、测序后,再克隆到原核表达载体pT7-PL上,转化大肠杆菌BL21(DE3 plys),以IPTG诱导大肠杆菌表达带有氨酸和人TREM-1蛋白组成的融合蛋白,经SDS-聚丙烯酰胺凝胶(PAGE)电泳初步鉴定,并对融合蛋白进行纯化。结果酶切电泳和DNA测序结果表明,成功地克隆了人TREM-1 eDNA;SDS-PAGE电泳结果显示相应分子质量37ku处有含人TREM-1融合蛋白的初步表达,证明了TREM-1原核表达的可行性。     

Use of loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. paratuberculosis

We evaluated the usefulness of loop-mediated isothermal amplification (LAMP) in detecting specific gene sequences of Mycobacterium avium subsp. paratuberculosis (MAP). A total of 102 primer sets for LAMP was designed to amplify the IS900, HspX, and F57 gene sequences of MAP. Using each of two primer sets (P-1 and P-2) derived from the IS900 fragment, it was possible to detect MAP in a manner similar to that used with nested PCR. The sensitivity of LAMP with P-1 was 0.5 pg/tube, which was more sensitive than nested PCR. When P-2 was used, 5 pg/tube could be detected, which was the same level of sensitivity as that for nested PCR. LAMP with P-1 was specific. Although only 2 Mycobacterium scrofulaceum strains out of 43 non-MAP mycobacterial strains were amplified, the amplification reaction for these strains was less efficient than for MAP strains, and their products could be distinguished from MAP products by restriction digestion. LAMP with P-2 resulted in very specific amplification only from MAP, the same result obtained with nested PCR. Our LAMP method was highly specific, and the white turbidity of magnesium pyrophosphate, a by-product of the LAMP reaction, allowed simple visual detection. Our method is rapid, taking only 2 h, compared with 4 h for nested PCR. In addition, the LAMP method is performed under isothermal conditions and no special apparatus is needed, which makes it more economical and practical than nested PCR or real-time PCR. These results indicate that LAMP can provide a rapid yet simple test for the detection of MAP.     

Loop-mediated isothermal amplification assay for the diagnosis of retinitis caused by herpes simplex virus-1

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of herpes simplex virus 1 (HSV-1). The specificity of the assay was tested using DNA extracted from HSV-1-infected rabbit corneal epithelium cultures, HSV-2 grown on Vero cell line, cytomegalovirus (CMV) (AD-169), varicella zoster virus (VZV) (Oka-vaccine), adenovirus, Aspergillus flavus and Staphylococcus aureus. The specificity of LAMP was confirmed by bidirectional sequencing of the amplicons. The sensitivity of the LAMP assay was tested using different concentrations of HSV-1 DNA. To evaluate the application of the LAMP assay in clinical diagnosis, we tested vitreous samples from 20 patients with suspected viral retinitis using LAMP and real-time PCR for HSV-1. The LAMP primers amplified only HSV-1 DNA; no LAMP products were detected with the DNAs of HSV-2, CMV, VZV, adenovirus A. flavus and S. aureus. The sequences of the positive HSV-1 LAMP products perfectly (99–100%) matched the HSV-1 sequences deposited in the GenBank database. LAMP is as sensitive as real-time PCR, with the lowest detection limit being 10 copies/μL of HSV-1 DNA. Of the 20 patients with suspected viral retinitis, four tested positive for HSV-1 using real- time PCR and LAMP. A 100% concordance was observed across the two methods. The LAMP assay is a rapid, highly specific and sensitive method for the diagnosis of retinitis caused by HSV-1.     

Sensitive and rapid detection of the new Delhi metallo-beta-lactamase gene by loop-mediated isothermal amplification

New Delhi metallo-β-lactamase 1 (NDM-1), which is associated with resistance to carbapenem, was first reported in 2008. A sensitive and rapid molecular assay to detect the plasmid bla(NDM-1) in clinical isolates is needed to control its spread. We describe a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of bla(NDM-1) from pure culture and sputum, urine, and fecal samples. Eight sets of primers were designed to recognize six or eight distinct sequences on target bla(NDM-1), and one set was selected as the most appropriate set of primers for its rapid detection. The specificity and sensitivity of the primers in the LAMP reactions for bla(NDM-1) detection were determined. The sensitivity of the LAMP assay for bla(NDM-1) detection in sputum, urine, and fecal samples was also tested. Two methods, namely, monitoring of turbidity and addition of calcein to the reaction tube, were used to determine negative and positive results. The results showed that target DNA was amplified and visualized by the two detection methods within 70 min at an isothermal temperature of 65°C. The sensitivity of LAMP, with a detection limit of 10.70 pg/μl DNA, was 100-fold greater than that of PCR. Thirteen infection bacterial strains without bla(NDM-1) were selected for testing of specificity, and the results of the amplification were negative, which showed that the primers had good levels of specificity. The LAMP method reported here is demonstrated to be a potentially valuable means for the detection of bla(NDM-1) and rapid clinical diagnosis, being fast, simple, and low in cost.     

Development of loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of ovine theileriosis in China

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid detection method in which the target deoxyribonucleic acid (DNA) can be efficiently amplified with high specificity and sensitivity under isothermal conditions using a set of either four or six specific primers. In this study, we have identified a conserved sequence for Theileria luwenshuni (UTRlu8) and for T. uilenbergi (UTRu6) suitable for designing a set of six primers for the simultaneous detection by LAMP of these pathogens causing theileriosis in sheep and goats in China. LAMP was performed at 63°C, and the amplified DNA was detectable within 15 min. The specificity of the reaction was confirmed through EcoRI restriction enzyme digestion analysis and sequencing. The assay was proven sensitive since specific amplification was obtained from 0.1 pg DNA of T. luwenshuni or T. uilenbergi. The LAMP assay was evaluated by testing 86 field samples in comparison to the reverse line blot method, showing a sensitivity and specificity of 66.0% and 97.4%, respectively. These results indicate that the LAMP assay is rapid and simple to run, cost effective, sensitive, and specific and has potential usefulness for application in diagnostics of and epidemiological studies on T. luwenshuni and T. uilenbergi infection of small ruminants.     

Comparison of in vitro culture and polymerase chain reaction for detection of Borrelia burgdorferi in tissue from experimentally infected animals.

A polymerase chain reaction (PCR) was developed for identification of Borrelia burgdorferi in biological specimens. The diagnostic efficiency was compared with that of in vitro culture. A primer set specifying a 791-bp DNA fragment of the B. burgdorferi B31 flagellin gene was used. Amplified DNA sequences were analyzed by agarose gel electrophoresis, and the identity of amplified DNA was confirmed by restriction enzyme cleavage and Southern blot hybridization with a 32P-labeled probe. By using purified B. burgdorferi DNA, the detection limit of the assay was approximately 0.002 pg of DNA, corresponding to one copy of the B. burgdorferi genome. By using in vitro-cultivated B. burgdorferi without prior DNA purification as the template DNA, 2 to 20 organisms could be detected. A 791-bp DNA fragment was amplified from all of 18 different B. burgdorferi strains tested, as well as from Borrelia hermsii and Borrelia anserina but not from Treponema pallidum. The efficacy of the PCR assay was evaluated on spleen, renal, and urinary bladder tissue specimens from eight experimentally infected gerbils. Specimens from the same organs were cultured in BSK medium in parallel. Of 24 organs, 21 (88%) were PCR positive and 17 (71%) were culture positive. All culture-positive specimens were also PCR positive. Compared with B. burgdorferi cultivation, PCR had at least a comparable diagnostic sensitivity, it was less laborious, and results were available within 1 to 2 days.     

Development of a loop-mediated isothermal amplification method for detection of Theileria lestoquardi

A loop-mediated isothermal amplification (LAMP) assay was developed for the diagnosis of Theileria lestoquardi infection. The primers were designed based on the clone-5 sequence of T. lestoquardi. The specificity and sensitivity of the assay were established. Analysis of the specificity showed that the selected LAMP primers amplified the target sequence from T. lestoquardi DNA successfully, while no amplification was seen with DNA from Theileria annulata, Theileria ovis, Babesia ovis, Anaplasma ovis, or ovine genomic DNA. The specificity of the LAMP product was further confirmed by restriction digestion and sequencing. The sensitivity of the LAMP assay was analyzed in comparison to PCR resulting in a detection limit of 10 fg/μl of plasmid DNA containing the clone-5 sequence. The suitability for utilizing the LAMP assay in the field for the diagnosis of T. lestoquardi infection was tested on 100 field samples collected in Sudan and compared with results obtained by PCR. The relative specificity and sensitivity of the established LAMP assay was determined to be 92.1% and 87.5%, respectively, indicating that it may be regarded as an alternative molecular diagnostic tool to PCR which could be used for epidemiological surveys on T. lestoquardi infection.     

Development and evaluation of a loop-mediated isothermal amplification method for the rapid detection of <Emphasis Type="Italic">Chlamydophila pneumoniae</Emphasis>

We developed a loop-mediated isothermal amplification (LAMP) method to detect Chlamydophila pneumoniae infection. This assay exclusively amplified C. pneumoniae sequences and no cross-reactivity was observed for other Chlamydia species. The detection limit for this assay was found to be ten elementary bodies in 25 min, as observed in a real-time turbidimeter and electrophoretic analysis. The specificity of the LAMP reaction was confirmed by restriction endonuclease analysis, as well as direct sequencing of the amplified product. Among nasopharyngeal swab specimens from 120 patients with acute respiratory tract infections and 40 healthy individuals, the LAMP results showed 100% agreement with the results of real-time polymerase chain reaction (PCR) assays.     

Quantitative detection of Streptococcus pneumoniae in nasopharyngeal secretions by real-time PCR

Streptococcus pneumoniae is an important cause of community-acquired pneumonia. However, in this setting the diagnostic sensitivity of blood cultures is below 30%. Since during such infections changes in the amounts of S. pneumoniae may also occur in the upper respiratory tract, quantification of these bacteria in nasopharnygeal secretions (NPSs) may offer a suitable diagnostic approach. Real-time PCR offers a sensitive, efficient, and routinely reproducible approach to quantification. Using primers and a fluorescent probe specific for the pneumolysin gene, we were able to detect DNA from serial dilutions of S. pneumoniae cells in which the quantities of DNA ranged from the amounts extracted from 1 to 10(6) cells. No difference was noted when the same DNA was mixed with DNA extracted from NPSs shown to be deficient of S. pneumoniae following culture, suggesting that this bacterium can be detected and accurately quantitated in clinical samples. DNAs from Haemophilus influenzae, Moraxella catarrhalis, or alpha-hemolytic streptococci other than S. pneumoniae were not amplified or were only weakly amplified when there were > or =10(6) cells per reaction mixture. When the assay was applied to NPSs from patients with respiratory tract infections, the assay performed with a sensitivity of 100% and a specificity of up to 96% compared to the culture results. The numbers of S. pneumoniae organisms detected by real-time PCR correlated with the numbers detected by semiquantitative cultures. A real-time PCR that targeted the pneumolysin gene provided a sensitive and reliable means for routine rapid detection and quantification of S. pneumoniae present in NPSs. This assay may serve as a tool to study changes in the amounts of S. pneumoniae during lower respiratory tract infections.     

Rapid, sensitive detection of Mycoplasma pneumoniae in simulated clinical specimens by DNA amplification.

The polymerase chain reaction (PCR) was investigated as a means of diagnosing Mycoplasma pneumoniae infections. The target DNA sequence was a 375-bp segment of the P1 virulence protein. This DNA segment was amplified in pure cultures of five different strains of M. pneumoniae but not in other species of Mycoplasma, Acholeplasma, or Ureaplasma that were tested. Simulated clinical specimens were used to compare PCR, culture, and the gene probe. The sensitivity of PCR was between 1 and 10 organisms. The sensitivity of culture was approximately 10(3) organisms, and the gene probe detected between 10(4) and 10(5) organisms. These results indicate that PCR has significant potential as a rapid, sensitive method for detecting M. pneumoniae in clinical specimens.