利用基因芯片快速检测多种临床常见致病菌及其耐药性基因

目的快速、准确、灵敏地检测临床常见的致病菌及其耐药基因。方法采用基因芯片技术，利用细菌23S rRNA基因序列信息和某些耐药基因作为检测靶基因，设计针对不同菌属的寡核苷酸探针和耐药基因探针的基因芯片，聚合酶链反应（PCR）扩增并荧光标记目的DNA片段，通过杂交反应检测致病菌及其耐药基因。结果在理想状态下（检测试剂盒抽提的细菌基因组DNA）检测的结果与国内外文献报道的灵敏度相当（10^3～10^6细菌／ml），并在部分临床分离株、耐药标准菌株中进行验证，显示该方法有良好的特异性及可重复性。细菌种类能鉴别到种水平的有16种，能鉴别到属水平的有7类。覆盖了临床常见的多种病原菌，并且还可同期检测超广谱β内酰胺酶（ESBLs）基因耐药。结论DNA芯片检测具有快速、高特异性和高灵敏度的特点，是常规鉴定方法的一种有益补充。     

Ultra-small-sample molecular structure detection using microslot waveguide nuclear spin resonance

We here report on the design of a planar microslot waveguide NMR probe with an induction element that can be fabricated at scales from centimeters to nanometers to allow analysis of biomolecules at nano- or picomole quantities, reducing the required amount of materials by several orders of magnitude. This device demonstrates the highest signal-to-noise ratio for a planar detector to date, measured by using the anomeric proton signal from a 15.6-nmol sample of sucrose. This probe had a linewidth of 1.1 Hz for pure water without susceptibility matching. Analysis of 1.57 nmol of ribonuclease-A shows high sensitivity in one- and two-dimensional NMR spectra. Along with reducing required sample volumes, this integrated geometry can be packed in parallel arrays and combined with microfluidic systems. Further development of this device may have broad implications not only for advancing our understanding of many intractable protein structures and their folding, molecular interactions, and dynamic behaviors, but also for high-sensitivity diagnosis of a number of protein conformational diseases.     

多重PCR快速检测恶性疟和间日疟的研究

目的 建立一种简便快速、能同时检测恶性疟和间日疟的核酸检测方法。方法 针对两种疟原虫18S rRNA基因设计2对(3条引物),优化引物浓度与退火温度,建立可扩增出两种疟原虫基因片段的多重PCR。并进行最低检测限确定和临床标本检测,以镜检法为金标准分析灵敏度和特异度等指标。结果 该方法可扩增出431 bp(恶性疟原虫)和341 bp(间日疟原虫)基因片段,最低检测限为10²copies/反应,检测临床标本的结果与镜检法无差别(P＞0.05),敏感度为93.55%,特异度为70.83%,阳性预测值为89.23%,阴性预测值为80.95%。结论 所建立的多重PCR方法可快速检测疟疾感染并鉴别分型,灵敏度高,值得推广。     

分子信标探针技术用于PCR检测诺卡氏菌SecA1基因

目的将诺卡氏菌属细菌的一段特异性DNA设计成分子信标探针,用于该细菌的PCR检测。方法将诺卡氏菌属细菌、戈登氏菌属细菌及红球菌属细菌菌株分别接种于脑心浸液琼脂培养基分离培养,观察其生长情况,提取菌株DNA作为扩增模板;设计诺卡氏菌属细菌基于secA1基因的特异性分子信标探针,在实时荧光定量PCR反应译体系中加入分子信标探针,PCR产物进行荧光信号检测。结果诺卡氏菌secA1基因经实时荧光定量PCR扩增后可产生阳性荧光信号,红球菌属细菌及戈登氏菌属细菌的secA1基因、阴性对照实验组及空白对照组经实时荧光定量PCR扩增后不产生荧光信号,为阴性。结论 secA1作为看家基因,是用来进行种水平的鉴定及系统进化研究非常理想的靶分子,而分子信标探针技术可以准确、快速、灵敏的进行诺卡氏菌secA1基因检测。     

大肠杆菌肠毒素基因多重PCR检测方法的建立

目的 建立一种快速检测大肠杆菌耐热肠毒素(heat-stable enterotoxin, STa, STb)和不耐热肠毒素(heat-labile enterotoxin, LT-Ⅰ, LT-Ⅱ)基因的多重PCR方法。方法 参照文献合成四对可扩增产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli, ETEC)耐热肠毒素基因(estA、estB)和不耐热肠毒素基因(elt-Ⅰ、elt-Ⅱ)的特异性引物,通过反应条件的优化,敏感性、特异性试验和临床样品检测,建立检测大肠杆菌肠毒素的多重PCR方法。结果 用所建立的多重PCR方法可特异性扩增出estA(229 bp)、estB(480 bp)、elt-Ⅰ(605 bp)和elt-Ⅱ(300 bp)基因片段,最低检出量分别为2.55×10¹ CFU/μL、2×10¹ CFU/μL、2×10¹ CFU/μL和2.47×10³ CFU/μL。从22株大肠杆菌分离株中检测到estA基因(2/22),elt-Ⅱ基因(3/22),未检测到estB和elt-Ⅰ基因,检测结果与常规PCR检测结果一致。结论 建立了检测大肠杆菌肠毒素基因(estA、estB、elt-Ⅰ和elt-Ⅱ)的多重PCR方法,该方法具有良好的特异性和敏感性,能够满足对细菌培养物的检测要求。     

Visualizing the dynamics of viral replication in living cells via Tat peptide delivery of nuclease-resistant molecular beacons

In this study, we describe the use of nuclease-resistant molecular beacons (MBs) for the real-time detection of coxsackievirus B6 replication in living Buffalo green monkey kidney (BGMK) cells via Tat peptide delivery. A nuclease-resistant MB containing 2′-O-methyl RNA bases with phosphorothioate internucleotide linkages was designed to specifically target an 18-bp 5′ noncoding region of the viral genome. For intracellular delivery, a cell-penetrating Tat peptide was conjugated to the MB by using a thiol–maleimide linkage. Presence of the Tat peptide enabled nearly 100% intracellular delivery within 15 min. When the conjugate was introduced into BGMK cell monolayers infected with coxsackievirus B6, a discernible fluorescence was observed at 30 min after infection, and as few as 1 infectious viral particle could be detected within 2 h. The stability and the intracellular delivery properties of the modified MBs enabled real-time monitoring of the cell-to-cell spreading of viral infection. These results suggest that the Tat-modified, nuclease-resistant MBs may be powerful tools for improving our understanding of the dynamic behavior of viral replication and for therapeutic studies of antiviral treatments.     

Biomedically important pathogenic fungi detection with volatile biomarkers

Volatile chemical profiles collected from the headspace of Aspergillus fumigatus (a pathogenic fungus that causes invasive pulmonary aspergillosis, allergic bronchopulmonary aspergillosis and chronic fungal sinusitis) grown on media with the connective tissue protein elastin, found in lung tissue, contained a large abundance of the sesquiterpene farnesene (3,7,11-trimethyl-1,3,6,10-dodecatetraene) and, depending on extraction time and sorbent material, other farnesene isomers and sesquiterpenes such as bisabolene (methyl-4-(6-methylhepta-1,5-dien-2-yl)cyclohex-1-ene). When human lung cells were cultured externally and infected with A. fumigatus, farnesene was also detected in each model lung system. Volatiles measured from cultured nasal lavage collected from a patient diagnosed with chronic fungal sinusitis, a condition frequently caused by A. fumigatus, revealed the presence of another pathogenic fungus, less frequently responsible for sinusitis, Epicoccum nigrum. The volatile profile of E. nigrum differed markedly from that of A. fumigatus with no sesquiterpenes detected.     

Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides

The development of a robust and portable biosensor for the detection of pathogenic bacteria could impact areas ranging from water-quality monitoring to testing of pharmaceutical products for bacterial contamination. Of particular interest are detectors that combine the natural specificity of biological recognition with sensitive, label-free sensors providing electronic readout. Evolution has tailored antimicrobial peptides to exhibit broad-spectrum activity against pathogenic bacteria, while retaining a high degree of robustness. Here, we report selective and sensitive detection of infectious agents via electronic detection based on antimicrobial peptide-functionalized microcapacitive electrode arrays. The semiselective antimicrobial peptide magainin I--which occurs naturally on the skin of African clawed frogs--was immobilized on gold microelectrodes via a C-terminal cysteine residue. Significantly, exposing the sensor to various concentrations of pathogenic Escherichia coli revealed detection limits of approximately 1 bacterium/μL, a clinically useful detection range. The peptide-microcapacitive hybrid device was further able to demonstrate both Gram-selective detection as well as interbacterial strain differentiation, while maintaining recognition capabilities toward pathogenic strains of E. coli and Salmonella. Finally, we report a simulated "water-sampling" chip, consisting of a microfluidic flow cell integrated onto the hybrid sensor, which demonstrates real-time on-chip monitoring of the interaction of E. coli cells with the antimicrobial peptides. The combination of robust, evolutionarily tailored peptides with electronic read-out monitoring electrodes may open exciting avenues in both fundamental studies of the interactions of bacteria with antimicrobial peptides, as well as the practical use of these devices as portable pathogen detectors.     

Multiplex allele specific PCR for rapid detection of extensively drug resistant tuberculosis

Effective tuberculosis (TB) control is hindered by lack of rapid diagnostic tests for detection of drug-resistant TB (DR-TB). Use of molecular tools for rapid detection of multi-and extensively- DR-TB, could facilitate early initiation of appropriate anti-tubercular treatment (ATT) regimen thereby interrupting transmission. Understanding the urgent situation, we standardized and evaluated 4 individual multiplex allele specific PCR (MAS-PCR) assays on 450 sputum specimens for Mycobacterium tuberculosis (MTB) detection and determination of drug resistance by targeting katG315, rpoB531, gyrA 94, rrs 1401 codon mutations for determination of resistance to Isoniazid (INH), Rifampicin (RIF), Fluoroquinolones (FQ) and Aminoglycosides (AG) respectively. Using a single sputum specimen, MAS-PCR correctly identified 97.2% (281/289; 95% CI:95-99) culture positive patients as MTB positive, 100% (271/271; 95% CI:99-100) for smear positive cases and 55.5% (10/18; 95% CI:34-75) for smear negative cases; and correctly identified 93.6% (104/111; 95% CI:87-97) of culture negative patients. Individual MAS-PCR assays reported variable diagnostic accuracy for determination of drug resistance. On comparison with phenotypic drug susceptibility testing, MAS-PCR assays correctly identified 89.2% (191/214; 95% CI:84-93), 94.9% (187/197; 95% CI:91-97), 72.5% (98/135; 95% CI:65-79) and 92.3% (24/26; 95% CI:75-99) of INH resistant, RIF resistant, FQ resistant and AG resistant specimens respectively; and correctly identified 94% (63/67; 95% CI:85-98), 86.9% (73/84; 95% CI:78-93), 93.1% (136/146; 95% CI:88-96) and 99.2% (253/255; 95% CI:97-100) of INH sensitive, RIF sensitive, FQ sensitive and AG sensitive specimens respectively. Thus, use of MAS-PCR assays for rapid detection of DR-TB is recommended, enabling early initiation of appropriate ATT.     

Immunosuppressive actions of retroviruses

The immunosuppressive properties of retroviruses were first demonstrated by Old et al. We later showed that Gross Passage A retrovirus superinfection in mice resulted in decreased antibody production and diminished allograft rejection. We have studied in some detail the immunosuppression which occurs subsequent to infection with feline leukemia virus (FeLV) as characterized by profoundly depressed T and B lymphocyte responses and decreased production of gamma-interferon. Injection of staphylococcal protein A (SPA) corrected these deficient immune responses, cleared circulating FeLV from blood and produced a regression of FeLV-induced lymphomas and leukemias. The immunosuppressive properties of FeLV and certain other retroviruses have been linked to the transmembrane viral envelope peptide, p15E. Cianciolo et al synthesized a 17-amino acid viral component which shares sequence homology with a highly conserved region of p15E. In vitro analyses have shown that this synthetic retroviral peptide suppresses T and B cell functions, inhibits the generation of cytotoxic lymphocyte (CTL) responses and dramatically alters the morphology and distribution of monocytes. The latter finding, along with reports that cells of the monocyte/macrophage lineage play a critical role in the initiation of human immunodeficiency infection, suggests that monocytes and macrophages may play a crucial role in retroviral infection and some of the associated immunodeficiencies associated with retroviral infection.     

马尔尼菲篮状菌致病机制相关分子研究进展

马尔尼菲篮状菌是一种重要的条件致病菌,主要流行于泰国、印度、中国大陆、中国香港、中国台湾、越南等亚洲热带地区。过往其主要感染人群为HIV患者,而近几年,非HIV患者感染在逐年增多。马尔尼菲篮状菌的致病性与形态的转变、黑色素的形成、生化代谢产物的产生等相关。近几年,有关马尔尼菲篮状菌致病机制,尤其是调控形态转化的基因有较深入的研究。本文对近年来马尔尼菲篮状菌相关毒力基因尤其是在代谢产物、色素形成、形态学转变上的调控基因进行的总结及评述。     

Development of Common Primer Multiplex PCR (CP-M-PCR) for species-specific meat detection

IntroductionA Common Primer Multiplex PCR (CP-M-PCR) was developed to detect three groups of animal species (swine, ruminant and pseudo-ruminant) from meat-based products. This method demonstrated a higher sensitivity and efficiency than the conventional multiplex PCR.ObjectiveTo develop a highly sensitive multiplex PCR method for rapid and accurate identification of meat in meat-based products.MethodsIn this study, a common forward primer was designed at a homologous region of mitochondrial NADH-dehyrogenase subunit 4 (Nad 4) gene sequences of all the animal groups. Adapter reverse primers were designed by adding an adapter sequence at the 5'-end of the specific reverse primers. PCR were performed on DNA extracted from muscle tissue samples using a common forward primer, adapter reverse primers and an adapter primer targeting sequence of Nad 4 gene. A serial of dilution of each reverse primer was used to determine and compare the sensitivity of CP-M-PCR to conventional multiplex PCR system. The detection limit of CP-M-PCR was evaluated with 10-fold serial dilutions of DNA concentration mixture in different ratios of concentration.Results & DiscussionThe use of adapter sequence at the 5'-end of the reverse primers increased the efficiency of the amplification and the application of a single forward primer solved the complexity in multiplex PCR system. Bands of specific amplification can be detected from the PCR assays containing as low as 10–6 μM of adapter reverse primer. This result indicated the sensitivity was tremendously increased as compared to the conventional multiplex PCR (10–3 μM). The limit of detection was as low as 1 ng of DNA.ConclusionCP-M-PCR has greatly improved the sensitivity and efficiency of the PCR system for detecting fraud in meat-based products, resulting in more reliable and accurate results than conventional multiplex PCR system.     

ETEC肠毒素基因多重PCR检测方法的建立

肠毒素性大肠杆菌的致病性与其具有粘附性的菌毛和产肠毒素的能力密切相关。由于菌毛的血清型多而复杂 ,肠毒素只有不耐热肠毒素 (LT)和耐热肠毒素 (ST)两种 ,因此成为研究的对象。用三对扩增产物分别为 1 1 0bp、2 37bp、368bp的引物建立了检测LT和STⅠ、STⅡ毒素基因的多重PCR方法。扩增产物分别用HindⅢ、HincⅡ、Sau3AⅠ限制性内切酶酶切 ,均得与预期一致的 2个片段。对各个参考株的检测结果为 1 0 0 %符合。结果表明该多重PCR方法具有很好的特异性和敏感性。该方法可用于肠毒素性大肠杆菌腹泻病的辅助诊断以及大肠杆菌的分类检测     

Multiplex ligation-dependent probe amplification for detection of genomic alterations in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is the commonest form of leukaemia in adults in Western countries. We performed multiplex ligation-dependent probe amplification (MLPA) analysis in 50 CLL patients to identify multiple genomic CLL-specific targets, including genes located at 13q14, 17p13 ( TP53 ), 11q23 ( ATM ) and chromosome 12, and compared the results with those obtained with fluorescence in situ hybridization (FISH). There was a good correlation between MLPA and FISH results, as most alterations (89%) were detected by both techniques. Only three cases with a low percentage (<25%) of cells carrying the alterations were not detected by MLPA. On the other hand, as MLPA uses multiple probes it identified intragenic or small alterations undetected by FISH in three cases. MLPA also detected alterations in 8q24 ( MYC ) and 6q25–26. In summary, unlike interphase FISH, MLPA enabled the simultaneous analysis of many samples with automated data processing at a low cost. Therefore, the combination of robust multiplexing and high throughput makes MLPA a useful technique for the analysis of genomic alterations in CLL.     

Multiplex polymerase chain reaction assay for selective detection of Salmonella enterica serovar typhimurium

A multiplex polymerase chain reaction (PCR) assay was developed for the identification of Salmonella enterica serovar Typhimurium. Three sets of primers were designed for detecting O4, H:i, and H:1,2 antigen genes from the antigen-specific genes rfbJ, fliC, and fljB, respectively. These were evaluated in a multiplex PCR assay by using DNAs from S. enterica serovar Typhimurium, 15 other Salmonella serovars, and 8 non-Salmonella enteric pathogens. Multiplex PCR proved to be capable of identifying S. enterica serovar Typhimurium specifically and differentiating it from other Salmonella serovars in addition to non-Salmonella enteric pathogens. Thus, this multiplex PCR assay can be practically applied to the identification of S. enterica serovar Typhimurium.