Development of a simple and sensitive technique for detection of point mutations in the K-ras oncogene

We sought to develop a simple and sensitive method based on mutant allele-specific amplification (MASA) for the detection of point mutations in the k-ras oncogene in blood samples. We used MASA and three nested MASA methods to detect a point mutation (GGT→GAT) in rat DHD cells at codon 12 of exon 1 of the k-ras gene. MASA allowed us to detect one k-ras mutated cell on a background of 10⁷ normal cells. The third nested-MASA (nested-MASA.c) method that we developed allowed us to detect one mutated cell among 10¹⁰ normal cells. Our methods should allow the detection of small amounts of mutant k-ras DNA in tissue, serum, and plasma, combining speed with efficiency and specificity.     

Rapid, sensitive and simple detection method for koi herpesvirus using loop-mediated isothermal amplification

New methods were developed for the detection of koi herpesvirus (KHV, CyHV-3) by LAMP, which were compared with the PCR for specificity and sensitivity. We designed two primer sets targeting a specific sequence within the 9/5 PCR amplicon (9/5 LAMP) and the upper region of the Sph I-5 PCR amplicon ( Sph I-5 LAMP), including a sequence highly conserved among the strains. The amplification was monitored in real-time based on the increase in turbidity, with magnesium pyrophosphate as the by-product. The reactions were carried out under isothermal conditions at 65°C for 60 min. The detection limit of both LAMP was six copies, equal to the modified Sph I-5 PCR. No cross-reactivity with other fish pathogenic viruses and bacteria was observed. Sph I-5 LAMP was found to have a quicker response in terms of the reaction velocity than 9/5 LAMP. Therefore, we consider Sph I-5 LAMP to be superior for routine use. Additionally, LAMP was found applicable to crude extract from gills and other organs. LAMP methods are superior in terms of sensitivity, specificity, rapidity and simplicity, and are potentially a valuable diagnostic tool for KHV infections.     

Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

Background  

Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment.     

Rapid and sensitive detection method of a bacterium by using a GFP reporter phage

A rapid, sensitive, and convenient method for detecting a specific bacterium was developed by using a GFP phage. Here we describe a model system that utilizes the temperate Escherichia coli-restricted bacteriophage lambda, which was genetically modified to express a reporter gene for GFP to identify the colon bacillus E. coli in the specimen. E. coli infected with GFP phage was detected by GFP fluorescence after 4-6 hr of incubation. The results show that a few bacteria in a specimen can be detected under fluorescence microscopy equipped with a sensitive cooled CCD camera. When E. coli and Mycobacterium smegmatis were mixed in a solution containing GFP phage, only E. coli was infected, indicating the specificity of this method. The method has the following advantages: 1) Bacteria from biological samples need not be purified unless they contain fluorescent impurities; 2) The infection of GFP phage to bacteria is specific; 3) The fluorescence of GFP within infected bacteria enables highly sensitive detection; 4) Exogenous substrates and cofactors are not required for fluorescence. Therefore this method is suitable for any phage-bacterium system when bacteria-specific phages are available.     

Rapid detection of antigen binding by antibody fragments expressed in the periplasm of Escherichia coli.

Bacterial expression systems can greatly facilitate protein engineering of antibodies. We have developed a system for high-level expression of antibodies, antibody fragments, or hybrid antibodies with novel effector functions in the periplasm of Escherichia coli. From 5 ml of cells, a simple extraction yields sufficient material for SDS-gel electrophoresis, detection and characterization of hapten binding. To demonstrate our system, heavy-chain variable regions and lambda 1 light chains of a mouse anti-NP antibody were synthesized as hybrid proteins with a bacterial signal peptide (Omp F). Each chain is secreted into the periplasm where processing (cleavage of the signal peptide), folding and heterodimer association take place. Periplasmic proteins are released by cold osmotic shock, and hapten-binding activity is easily detected without further manipulation. The ease of genetic engineering in this system will facilitate the production of immunoglobulin derivatives designed for specific applications, and expression of these molecules in a native state will allow the rapid screening of combinatorial libraries and the results of mutagenesis.     

Rapid antibody screening by membrane chromatographic immunoassay technique

This paper describes a novel membrane chromatographic immunoassay technique suitable for rapid screening of antibodies in serum samples. This technique could potentially be utilized for antibody screening in situations where screening for exposure to one of several possible antigens is required. A synthetic microporous membrane is first selectively loaded with antibodies from the test serum sample by hydrophobic interaction. The in situ affinity membrane thus formed is sequentially pulsed with the antigens corresponding to the antibodies being screened. From the antigen chromatogram peak profile thus obtained, inferences about the antibodies present in the serum sample can then easily be made. This technique in addition to being rapid and direct is conceptually simple, and does not use any expensive media or reagents. It would potentially be useful for rapid diagnosis of infections as well as for rapid assessment of conditions such as envenomation or exposure to toxic substances.     

Highly sensitive and rapid detection of antibody catalysis by luminescent bacteria

A highly sensitive, inexpensive, and facile bioluminescent assay for the detection of catalytic antibodies has been developed. This assay may be used for the early detection of antibody catalysis. The efficiency of this technique was exemplified by the use of the luminescent bacterium VhM42 for monitoring an antibody-catalyzed retroaldol fragmentation reaction with aldolase antibodies 38C2 and 24H6.     

Rapid and sensitive detection of retrovirus entry by using a novel luciferase-based content-mixing assay

We describe a novel assay that permits measurement of entry of murine leukemia virus and pseudotypes with greater sensitivity and more rapidly than previously possible. To achieve this, we encapsulated a sensitive reporter enzyme, luciferase, directly into fully infectious, intact viral particles. The enzyme is specifically targeted to the viral lumen, as a C-terminal fusion on the viral envelope protein. Only when the incorporated luciferase is released from the viral lumen and gains access to its substrates is light emitted and readily detected. When cells are perfused with luciferin, quantitative measurements of entry can be made in real time on live cells. Uniquely, the amount of cell-bound virus can be determined in the same assay by addition of detergent to expose the luciferase. We demonstrate that virus carrying a mutation in the fusion peptide binds normally to cells but is unable to infect them and gives no entry signal. Using this assay, we show that inhibitors of endosomal acidification inhibit signal from vesicular stomatitis virus pseudotypes but not murine leukemia virus, consistent with a pH-independent mode of entry for the latter virus. Additionally, the fusion kinetics are rapid, with a half-life of 25 min after a delay of 10 to 15 min. The future use of this assay will permit a detailed examination of the entry mechanism of viruses and provide a convenient platform to discover novel entry inhibitors. The design also permits packaging of potential therapeutic protein cargoes into functional virus particles and their specific delivery to cellular targets.     

基于量子点标记的赭曲霉毒素A快速、高灵敏荧光免疫层析检测方法的建立及应用

赭曲霉毒素A（ochratoxin A,OTA）具有肾毒性、致畸性、致癌性和免疫毒性,广泛存在于各种粮食作物及其副产品中,是食品和饲料原料的重要污染物,可在人类及动物体内蓄积,在已知发现的真菌毒素中,重要性和危害性仅次于黄曲霉毒素。本研究通过采用量子点荧光微球（quantum dots,QDs）标记OTA单克隆抗体,并基于免疫层析原理,优化、建立了OTA高灵敏荧光免疫层析检测方法（FICGA）,15min即可实现对农产品中OTA污染的快速定量检测。该方法检测下限（IC₁₀）达到0.04ng/mL,检测区间（IC₂₀-IC₈₀）为0.05-0.59ng/mL,半数抑制率（IC₅₀）为0.18ng/mL。与OTA类似物OTB、OTC交叉反应性为7.3%和11.9%,对其他常见真菌毒素AFB₁、ZEN、FB₁和DON均无交叉反应。在玉米、面粉和大豆样本中的加标回收率可达83.2%-117.8%,与LC-MS/MS同时对天然样本中OTA含量的检测结果表明,两种方法相关性良好。本研究建立的FICGA快速、灵敏,可满足基层单位和现场的快速检测需求,具有很好的应用前景。     

Rapid and sensitive detection by PCR of mpcI gene in the rhizosphere

Abstract: A simple and sensitive detection system, using polymerase chain reaction (PCR) and a soil microcosm, was developed to detect a bacterial catabolic gene in the rhizosphere. The inoculated population of Alcaligenes eutrophus JMP134, a phenol and 2,4-dichlorophenoxy acetic acid utilizer, was readily detected by this technique, which permitted taking of samples from specific locations of root (including rhizosphere) and soil. The number of JMP134 viable cells (10²–10³ cells), typically picked up by the nitrocellulose filter strip method, yielded sufficient amount of the target DNA to be detected by PCR. Primers encoding metapyrocatechase I (MPC I; catechol 2,3-dioxygenase) enabled the discrimination of at least five viable cells of JMP134 among the indigenous microorganisms inhabiting bush bean roots. This simplified PCR detection procedure facilitated monitoring of the specific degradative gene in the rhizosphere in only 5 h.     

Rapid and sensitive detection of b-galactosidase-producing yeasts by using microtiter plate assay

Yeast b-galactosidase activity was detected by a microtiter plate assay using pNPG or Xgal as substrate in 30 minutes. The detection gave a clear result which is well correlated with the specific b-galactosidase activity present in each strain studied. The microtiter plate assay is an effective method to improve the detection and quantify the b-galactosidase gene in recombinant strains of Saccharomyces cerevisiae.     

Rapid and sensitive detection of Singapore grouper iridovirus by loop-mediated isothermal amplification

Aims:  The aim of this paper was to develop a loop-mediated isothermal amplification (LAMP) method for rapid, sensitive and inexpensive detection of Singapore grouper iridovirus (SGIV) in grouper (GP), Epinephelus sp.
Methods and Results:  A set of six specific primers was designed by targeting the SGIV ORF-014L. With Bst DNA polymerase large fragment, the target DNA can be amplified as early as 20 min at 65°C in a simple water bath. The detection limit is about 0·02 fg (equivalent to 6·3 copies) of plasmid ORF-014L. LAMP products could be judged with three different methods. There were no cross-reactions with seven other aquatic animal viruses indicating high specificity of the LAMP. The LAMP method was applied to detect SGIV in virus-infected GP cells and GP tissues effectively.
Conclusions:  The LAMP described in this study is a cheap, sensitive, specific and rapid protocol for the detection of SGIV in cells and in GP tissues.
Significance and Impact of the Study:  The developed LAMP method can be simply applied both in field condition and in laboratory operation for specific detection of SGIV infection.     

Detection of elastin by immunoelectronmicroscopy

Summary By applying a double-immunolabeling technique to preembedded tissue preparations, we demonstrated the existence of serotoninergic innervation to neurons containing vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus (SCN). Immunoreactivity for serotonin and VIP was revealed by the presence of diaminobenzidine (DAB) reaction products and silver-intensified DAB reaction products, respectively; in a further stage, the silver grains were substituted with gold particles. DAB reaction products were precipitated on the surface of vesicular structures, while gold particles were scattered densities. Serotoninergic axons were numerous and closely packed together, occasionally forming synaptic junctions with gold-labeled VIP-containing neurons. At these synaptic junctions, small vesicular structures accumulated to form a coat under the presynaptie membrane, and the postsynaptic membrane was lined with a homogeneous accumulation of fine deposits. This postsynaptic apparatus varied in appearance; some parts were flat and thin, while others were of irregular thickness. Serotoninergic fibers also formed synaptic junctions with unidentified neurons, in which postsynaptic membrane specialization was also observable. As VIP-containing neurons are known to be synapsed by somatostatin (SRIH)-containing neurons, their regulation must involve both serotonin and SRIH at least.     

Rapid detection of members of the family Enterobacteriaceae by a monoclonal antibody.

Six monoclonal antibodies directed against enterobacteria were produced and characterized. The specificity of one of these antibodies (CX9/15; immunoglobulin G2a) was studied by indirect immunofluorescence against 259 enterobacterial strains and 125 other gram-negative bacteria. All of the enterobacteria were specifically recognized, the only exception being Erwinia chrysanthemi (one strain tested). Bacteria not belonging to members of the family Enterobacteriaceae were not detected, except for Plesiomonas shigelloides (two strains tested), Aeromonas hydrophila (five strains tested), and Aeromonas sobria (one strain tested). This recognition spectrum strongly suggested that CX9/15 recognized the enterobacterial common antigen. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) experiments, the six antienterobacteria antibodies presented similar specificities; they all revealed only one band with an apparent molecular weight of about 20,000 from the crude extract of an enterobacterium. The six monoclonal antibodies, and especially CX9/15, can be used to develop new tests for rapid and specific detection of enterobacteria.     

Rapid detection of members of the family Enterobacteriaceae by a monoclonal antibody.

Six monoclonal antibodies directed against enterobacteria were produced and characterized. The specificity of one of these antibodies (CX9/15; immunoglobulin G2a) was studied by indirect immunofluorescence against 259 enterobacterial strains and 125 other gram-negative bacteria. All of the enterobacteria were specifically recognized, the only exception being Erwinia chrysanthemi (one strain tested). Bacteria not belonging to members of the family Enterobacteriaceae were not detected, except for Plesiomonas shigelloides (two strains tested), Aeromonas hydrophila (five strains tested), and Aeromonas sobria (one strain tested). This recognition spectrum strongly suggested that CX9/15 recognized the enterobacterial common antigen. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) experiments, the six antienterobacteria antibodies presented similar specificities; they all revealed only one band with an apparent molecular weight of about 20,000 from the crude extract of an enterobacterium. The six monoclonal antibodies, and especially CX9/15, can be used to develop new tests for rapid and specific detection of enterobacteria.