A rapid method for detecting fungi-toxic substances

A simple and rapid technique is reported for the preliminary screening of fungi-toxic extracts/samples by direct spotting onto silica gel plates and subsequent over-spraying with a fungal spore suspension. After incubation fungi-toxicity is indicated by a growth inhibition zone, the area of which is related to the concentration of the sample.B.K. Rana and V. Taneja are with the Department of Biochemistry. Faculty of Science, Banaras Hindu University, Varanasi 221005, India; U.P. Singh is with the Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005, India.     

一种快速提取基因组DNA的方法

采采用氧化硅超顺磁性纳米磁珠和自己设计的试剂体系及提取流程,建立了一种基因组DNA的快速提取方法,该方法以氧化硅磁珠为固相吸附载体,盐酸胍、 -巯基乙醇和SDS为主要裂解吸附试剂。以全血或培养细胞为实验材料进行基因组DNA的提取结果显示用本文建立的方法提取100 L小鼠抗凝血,可得2～3 g基因组DNA, OD260/OD280为1.8 ± 0.05,其纯度可满足后续的酶切和PCR生物操作要求。该方法整个提取过程只需12分钟,不需特殊实验条件同时可省略蛋白酶K的消化过程和离心操作,适用于一般实验室的需求,是一种操作简便、快速高效的提取方法。     

A rapid method for detecting malformations in rat fetuses

A rapid method for examining rat fetuses is presented. The technique consists of fixing the fetuses in Bouin's solution, serially sectioning the head, neck and lower trunk with a razor blade and doing sagittal sections of the heart after opening the thoracic cavity. Examples of sections from normal 20 day rat fetuses are given as well as some with the following abnormalities: cleft palate produced by chlorcyclizine and eye and heart malformations resulting from anti-adult rat kidney serum.     

A rapid method for producing highly purified Cryptosporidium parvum oocysts

Most procedures that have been described for purifying Cryptosporidium parvum oocysts are designed to either identify the parasites in clinical specimens or isolate oocysts from a small volume of feces from infected animals. The present study describes a rapid method for purifying high numbers of C. parvum oocysts from feces of infected calves that contains minimal contaminating fecal material and bacteria. The isolation method is based on differential flotation of C. parvum oocysts in NaCl, followed by ether extraction to solubilize lipids in calf feces. This procedure regularly yields > 10(9) purified C. parvum oocysts within 1-2 days of feces collection.     

A rapid method for protein extraction from fission yeast

Researchers working with fission yeast conduct protein extraction widely and frequently, but this includes the handling of glass beads, and hence is laborious and cumbersome, especially when dealing with a large number of samples. Here we describe a rapid and reliable method for preparing protein extract from fission yeast, one which is applicable to routine western blotting.     

A rapid plate culture method for screening of amylase producing micro-organisms

A simple and rapid method is described for the detection of amylase-producing micro-organisms on solid media using Remazol Brilliant Blue-starch as substrate. The blue starch is incorporated into nutrient agar and amylase production is detected by the disappearance of the colour around the colonies. The method which is non-destructive, allows for direct visualization and isolation of amylolytic micro-organisms from the environment without prior replication of colonies.     

Sterols and fatty acids of aflatoxin and non-aflatoxin producing isolates of Aspergillus

The fatty acids and sterols present in 5 isolates of Aspergillus flavus and 3 isolates of A. parasiticus were determined; 2 isolates within each species were aflatoxin producers. The 4 major fatty acids were 16:0, 18:0, 18:1 and 18:2 with a trace of 15:0 in one isolate and traces of 17:0 in 3 other isolates. Cholesterol, ergosterol and 5, 7-ergostadienol were present in all isolates; the 5 isolates of A. flavus could be identified on the basis of retention times of minor sterols present. There was no correlation of total lipids, fatty acids or sterols with the production of aflatoxins. Water soluble complexes of sterols were not detected.     

A new method for rapid identification of influenza virus isolates.

With the use of bacteria sensitized by influenza virus strain-specific antisera, virus isolates can be identified rapidly. One drop of virus suspension is mixed with one drop of sensitized bacteria on a slide that is then agitated; reaction occurs within 10 minutes. The test is subtype-specific. The mehod is based on the fact that the cell wall of the Cowan type 1 strain of Staphylococcus aureus contains abundant quantities of an antigen, known as protein A, that reacts with the IgG molecule by binding it in such a manner that the antibody-combining sites remain free. If an antigen homologous to the antibody coated on the surface of the bacteria is added to the suspension of sensitized staphylococci, agglutination occurs.     

A rapid DNA extraction method for sugarcane and its relatives

A simple DNA extraction method based on CTAB precipitation was used to obtain DNA from members of the genusSaccharum and related species. DNA yields and purities were similar for allSaccharum species sampled. The method described here resulted in high quality total DNA suitable for polymerase chain reaction (PCR)-based techniques as well as restriction endonuclease digestion, Southern hybridization, and DNA cycle-sequencing.     

A microengraving method for rapid selection of single cells producing antigen-specific antibodies

Monoclonal antibodies that recognize specific antigens of interest are used as therapeutic agents and as tools for biomedical research. Discovering a single monoclonal antibody requires retrieval of an individual hybridoma from polyclonal mixtures of cells producing antibodies with a variety of specificities. The time required to isolate hybridomas by a limiting serial-dilution, however, has restricted the diversity and breadth of available antibodies. Here we present a soft lithographic method based on intaglio printing to generate microarrays comprising the secreted products of single cells. These engraved arrays enable a rapid (<12 h) and high-throughput (>100,000 individual cells) system for identification, recovery and clonal expansion of cells producing antigen-specific antibodies. This method can be adapted, in principle, to detect any secreted product in a multiplexed manner.     

A rapid column-based ancient DNA extraction method for increased sample throughput

Genetic analyses using museum specimens and ancient DNA from fossil samples are becoming increasingly important in phylogenetic and especially population genetic studies. Recent progress in ancient DNA sequencing technologies has substantially increased DNA sequence yields and, in combination with barcoding methods, has enabled large-scale studies using any type of DNA. Moreover, more and more studies now use nuclear DNA sequences in addition to mitochondrial ones. Unfortunately, nuclear DNA is, due to its much lower copy number in living cells compared to mitochondrial DNA, much more difficult to obtain from low-quality samples. Therefore, a DNA extraction method that optimizes DNA yields from low-quality samples and at the same time allows processing many samples within a short time frame is immediately required. In fact, the major bottleneck in the analysis process using samples containing low amounts of degraded DNA now lies in the extraction of samples, as column-based methods using commercial kits are fast but have proven to give very low yields, while more efficient methods are generally very time-consuming. Here, we present a method that combines the high DNA yield of batch-based silica extraction with the time-efficiency of column-based methods. Our results on Pleistocene cave bear samples show that DNA yields are quantitatively comparable, and in fact even slightly better than with silica batch extraction, while at the same time the number of samples that can conveniently be processed in parallel increases and both bench time and costs decrease using this method. Thus, this method is suited for harvesting the power of high-throughput sequencing using the DNA preserved in the millions of paleontological and museums specimens.     

A rapid non-destructive DNA extraction method for insects and other arthropods

Preparation of arthropods for morphological identification often damages or destroys DNA within the specimen. Conversely, DNA extraction methods often destroy the external physical characteristics essential for morphological identification. We have developed a rapid, simple and non-destructive DNA extraction technique for arthropod specimens. This technique was tested on four arthropod orders, using specimens that were fresh, preserved by air drying, stored in ethanol, or collected with sticky or propylene glycol traps. The technique could be completed in 20 min for Coleoptera, Diptera and Hemiptera, and 2 min for the subclass Acarina, without significant distortion, discolouration, or other damage to the specimens.     

FTA-DNA直接提取法在病原真菌分子鉴定中的应用

目的建立并评价FTA-DNA直接提取法在病原真菌分子鉴定中的应用。方法采用whatman FTA-DNA直接提取法从25个不同种属的45株培养的菌株和6例临床标本中提取病原真菌DNA,用于病原真菌的测序鉴定。配制不同浓度的孢子悬液探索该方法的检测限和安全性。结果 45株菌株扩增后均能得到1条清晰的DNA扩增片段,并成功测序。应用该方法亦成功从腹水、胸水、口腔拭子、宫颈拭子来源的临床标本中直接提取DNA并成功鉴定病原真菌。该DNA提取方法联合降落PCR能检测到1.0×103个cell/mL的孢子悬液,1.0×104个cell/mL及以下浓度的孢子悬液可以被FTA卡完全灭活。结论 FTA-DNA直接提取法可快速有效地从培养的菌株及部分临床标本中提取并保存病原真菌DNA,用于病原真菌的测序鉴定。     

A rapid DNA extraction method for PCR amplification from wetland soils

Aims: We tested a method of rapid DNA extraction from wetland soil samples for use in the polymerase chain reaction. Methods and Results: The glass bead/calcium chloride/SDS method obtained in the present study was compared with the calcium chloride/SDS/enzymatic extraction method and the UltraClean? Soil DNA Isolation Kit. Rapid DNA extraction could be completed within about two hours without purification steps. Conclusions: This study succeeded in establishing a fast soil DNA extraction protocol that can be applied to various environmental sources that are rich in humic acid content. Significance and Impact of the Study: The method provides a technology with high‐quality DNA extraction from soils for testing the diversity of AOB and AOA.     

A rapid method for detecting specific amplified PCR fragments in microtiter plates.

A simple method is presented to circumvent laborious and time consuming electrophoretic separations of specific PCR amplification products. Specific target DNA is amplified using nucleotides labelled with DIG-dUTP or biotin-dCTP. The labelled PCR products are separated from unincorporated nucleotides or oligonucleotides by using a positively charged DEAE cellulose matrix. Amplification products are visualized directly in the matrix using immunoenzymatic methods or streptavidin-conjugated enzymes. The detection process can be carried out within 2 h, allows the processing of large sample sizes and can potentially be automated.     

A rapid method for extraction of zearalenone and zeraralenols in fermented corn

A rapid extraction method is described for isolation of zearalenone and α- and β-trans-zearalenols from laboratory fermented corn. Corn fermented withFusarium crookwellense at 25°C for 2 weeks was agitated for 5 minutes in acetone. The acetone extract was evaporated to dryness and the remaining residue was chromatographed on a silica gel column with hexane:ethyl acetate (8:2). The products eluted with the hexane:ethyl acetate mixture in 1–1/2 column volumes and were identified by regular phase and C-18 reverse-phase thin layer chromatography. The products were verified by gas chromatography-mass spectroscopy. Product recoveries were 62.5–70% for the zearalenols and 70% for zearalenone in the range 0.5–50 mg/Kg.