PCR-based quantification of Pneumocystis carinii in in vitro systems

In many laboratories, PCR has become a routine method for the sensitive diagnosis of Pneumocystis carinii in patient samples. In contrast, quantification of fungal numbers in in vitro setups still largely relies on more conventional procedures such as histological stainings. These are time consuming and their applications are limited when dealing with small fungal numbers contaminated with tissue and cellular debris. This study presents a sensitive and rapid method for P. carinii quantification based on PCR analysis that can be easily integrated into standard detection procedures without requiring any major additional steps. P. carinii-specific PCR performed with total DNA extracted from both standard samples with known fungal numbers and experimental samples was quantified relative to PCR products of a standard concentration from a control plasmid added prior to DNA extraction. This measure controlled for variations in DNA extraction and PCR efficiency among the samples to be compared. The correlation between analyzed P. carinii-specific DNA and the actual fungal numbers employed was highly significant.     

Rapid differentiation and enumeration of the total, viable vegetative cell and spore content of thermophilic bacilli in milk powders with reference to Anoxybacillus flavithermus

AIMS: The development of a rapid method for the selective detection and enumeration of the total and viable vegetative cell and spore content of thermophilic bacilli in milk powder by PCR. METHODS AND RESULTS: Quantitative PCR and microscopy indicate the presence of up to 2.9 log units more cells in milk powder than accounted for by plate counting due to the majority of cells being killed during milk processing. Two approaches for viable and dead cell differentiation of thermophilic bacilli by quantitative PCR were evaluated, these being the nucleic binding dye ethidium monoazide (EMA) and DNase I digestion. The former agent exposed to a viable culture of Anoxybacillus flavithermus caused considerable cell inactivation. In contrast, DNase I treatment had no effect on cell viability and was utilized to develop DNA extraction methods for the differential enumeration of total, viable vegetative cells and spores in milk powder. Moreover, the methods were further applied and evaluated to 41 factory powder samples taken throughout eight process runs to assess changes in numbers of vegetative cells and spores with time. DNase I treatment reduced vegetative cell numbers enumerated with PCR by up to 2.6 log units. The quantification of spores in the factory milk powders investigated indicates on average the presence of 1.2 log units more spores than determined by plate counting. CONCLUSIONS: The method presented in this study provides the ability to selectively enumerate the total and viable cell and spore content of reconstituted milk. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study provides a tool to monitor the extent of thermophilic contamination during milk powder manufacturing 60-90 min after sampling.     

Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using real-time PCR analysis

Three comparatively rapid methods for the extraction of DNA from fungal conidia and yeast cells in environmental (air, water and dust) samples were evaluated for use in real-time PCR (TaqMan™) analyses. A simple bead milling method was developed to provide sensitive, accurate and precise quantification of target organisms in air and water (tap and surface) samples. However, quantitative analysis of dust samples required further purification of the extracted DNA by a streamlined silica adsorption procedure.     

Quantitative detection of messenger RNA by solution hybridization and enzyme immunoassay

A novel nucleic acid detection technique is described for the quantitative measurement of eukaryotic mRNA in biological samples. The procedure involves two steps: a hybridization reaction in solution with a biotinylated cDNA probe, and a conventional enzyme immunoassay that uses a monoclonal antibody for DNA.RNA hybrids to detect the specific mRNA.cDNA complexes. The method has comparable sensitivity to 32P-based methods and yields results that are quantitative and highly reproducible. Furthermore, the test can be performed using unfractionated cytoplasm without the need for extraction with organic solvents. This technique provides a rapid and quantitative method for studying changes in cellular mRNA levels, and it is suitable for testing large numbers of samples.     

Relevance of Cryptosporidium parvum hsp70 mRNA amplification as a tool to discriminate between viable and dead oocysts

Two mRNA extraction methods were compared in this study to clarify the discrepancies found between authors regarding the presence of mRNA in inactivated Cryptosporidium parvum oocysts. Cryptosporidium parvum heat shock protein 70 (hsp70) mRNA extraction was performed by using oligo(dT)20-labeled magnetic beads or by incubating oocyst lysates with DNase I. Significant differences in mRNA recovery rates between these 2 techniques were observed when working on inactivated oocysts. We consistently detected hsp70 mRNA in oocysts heated at 60 C for 30 min and oocysts incubated in 10% formalin for 2 hr when using DNase I in the mRNA extraction procedure. In contrast, no mRNA was detected in such oocysts when magnetic beads were used for the mRNA extraction. The selective capture of long poly-A tail mRNA, when using oligo(dT)20-labeled magnetic beads, is proposed in this paper for explaining the discrepancies observed between the two mRNA extraction methods compared in this study. DNA decay in inactivated and aging oocysts makes quantitative polymerase chain reaction a potential alternative technique for assessing C. parvum oocyst viability status in environmental samples.     

Extraction of DNA from soil using nanoparticles by magnetic bioseparation

Aims: To develop a simple, rapid and inexpensive soil DNA extraction protocol. Methods and Results: The protocol relies on the use of superparamagnetic silica‐magnetite nanoparticles for the isolation and purification of DNA from soil samples. DNA suitable for use in molecular biology applications was obtained from a number of soil samples. Conclusions: The DNA extracted using the tested method successfully permitted the PCR amplification of a fragment of the bacterial 16S rDNA gene. The extracted DNA could also be restriction endonuclease digested. Significance and Impact of the Study: The protocol reported here is simple and permits rapid isolation of PCR‐ready soil DNA. The method requires only small quantities of soil sample, is scalable and suitable for automation.     

Rapid and Reliable Differential Display from Minute Amounts of Tissue: Mass Cloning and Characterization of Differentially Expressed Genes from Loblolly Pine Embryos

Performing RNA differential display analysis on small tissue samples is difficult since much RNA, the initial template for the reaction, is lost during conventional isolation procedures. We have developed a rapid method which employs oligo-dT beads to capture mRNA from cell lysates. Subsequent reactions are primed directly from the beads, thus RT and PCR reactions can be completed within a few hours of tissue harvest. This approach allows us to perform differential display on a single pine embryo. We describe strategies for distinguishing classes of co-migrating bands excised from differential display gels and outline a PCR-based method for confirming differential expression of large numbers of cloned bands in cases where RNA quantities are limiting.     

Use of the polymerase chain reaction and 16S rRNA sequences for the rapid detection of Brochothrix spp. in foods

Oligonucleotide primers were designed against rRNA sequences to give a DNA-based PCR assay for the rapid identification/detection of Brochothrix spp. The PCR products could be confirmed by hybridization to an internal oligonucleotide probe. The method successfully and sensitively detected/identified these organisms in pure cultures but was of limited value as a detection method because the detection sensitivity, in relation to conventional plate counts, varied and the assay sensitivity was reduced in the presence of staphylococci. Furthermore, sensitivity was also lost when the assay was applied directly to meat samples. However, a separation step using a lectin (from Agaricus bisporus ) immobilized on magnetic beads prior to the PCR assay, allowed the direct detection of low numbers (> 10 cfu g^-1) of Brochothrix in meat samples within a working day.     

基于功能化磁珠快速提取植物病毒RNA方法的建立与优化

植物病毒病是制约作物生产的主要病害之一。及时明确其病毒病原和发展规律是有效控制其大规模传播的前提。而现有植物病毒病检测技术存在周期长、步骤繁琐、检测环境严苛等缺点。本研究以烟草花叶病毒 (Tobacco mosaic virus,TMV)为模型,基于碱基互补配对原则设计针对TMV的功能化磁珠（CMBs-ACPTMV）进行RNA提取,并对功能化磁珠的制备条件、提取反应条件以及灵敏性和稳定性等性能进行优化分析。结果表明,当添加4 μmol捕获探针（ACPTMV）、0.08 mg羧基磁珠（CMBs）时,所制备的CMBs-ACPTMV吸附RNA的能力最好;当提取时间为3 min时,CMBs-ACPTMV提取RNA的效果最好,而改变CMBs-ACPTMV的提取温度时其提取能力无明显变化;性能评价分析发现,CMBs-ACPTMV的灵敏度可达2.5 ng/μL,且检测稳定性较好。与常规RNA提取技术相比,CMBs-ACPTMV在检测时间和样品消耗量上具有突出优势。本研究所建立的功能化磁珠提取法快速、安全和简便,只需简易设备便可实现植物病毒RNA的快速提取,具有广阔的应用前景。     

免疫胶体金法提取环境标本中细菌DNA技术

将抗-DNA单克隆抗体标记在胶体金颗粒上制成免疫胶体金试剂,提取标本中DNA,直接用于PCR检测,从而建立一种简单、快速、高效的免疫胶体金方法提取环境标本中的DNA。结果表明：应用免疫胶体金试剂可有效去除环境标本中PCR抑制剂,浓缩模板,提高PCR检测敏感度3～4个数量级。操作步骤简单,无需使用有机溶剂,避免环境污染,吸附了DNA的免疫胶体金可直接用于PCR扩增。研制了免疫胶体金试剂并确定其最佳反应条件,有效提高PCR技术在检测现场环境标本中的敏感性和实用性。     

Towards an international standard for PCR-based detection of foodborne thermotolerant campylobacters: interaction of enrichment media and pre-PCR treatment on carcass rinse samples

As part of a large EU project for standardisation of polymerase chain reaction (PCR), a systematic evaluation of the interaction of enrichment media, type of DNA polymerase and pre-PCR sample treatment for a PCR detecting thermotolerant campylobacters was carried out. The growth-supporting capacity and PCR compatibility of enrichment in Preston, Mueller-Hinton and Bolton broth (blood-containing and blood-free) were evaluated. The effect of resin-based DNA extraction and DNA extraction by boiling on the final PCR assay was investigated. The time-course studies indicated that a 20-h sample enrichment in blood-containing Bolton broth, followed by a simple resin-based extraction of DNA and a PCR amplification using Tth polymerase, resulted in strong and clear PCR amplicons for target (287 bp) and internal amplification control (IAC, 124 bp). The enrichment PCR-based method, tested on 68 presumably naturally contaminated poultry-rinse samples, showed a diagnostic sensitivity of 97.5% (39 PCR-positive/40 total positive samples) and a diagnostic specificity of 100% (28 PCR-negative/28 total negative samples; P=0.32) when compared to a standard bacteriological method (ISO 10272).     

LightCycler real-time PCR for rapid detection and quantitation of Mycobacterium leprae in skin specimens

Diagnosis of leprosy is usually based on clinical features and skin smear results including the number of skin lesions. Mycobacterium leprae is not cultivable and bacterial enumeration by microscopic examination is required for leprosy classification, choice in choosing and monitoring chemotherapy regimens, and diagnosis of relapse. However, detection and quantification using standard microscopy yields results of limited specificity and sensitivity. We describe an extremely sensitive and specific assay for the detection and quantification of M. leprae in skin biopsy specimens. Primers that amplified a specific 171-bp fragment of M. leprae 16S rRNA gene were chosen and specificity was verified by amplicon melting temperature. The method is sensitive enough to detect as low as 20 fg of M. leprae DNA, equivalent to four bacilli. The assay showed 100% concordance with clinical diagnosis in cases of multibacillary patients, and 50% of paucibacillary leprosy. The entire procedure of DNA extraction and PCR could be performed in c. 3 h. According to normalized quantitative real-time PCR, the patients in this study had bacilli numbers in the range of 1.07 x 10(2) -1.65 x 10(8) per 6-mm3 skin biopsy specimen. This simple real-time PCR assay is a facile tool with possible applications for rapid detection and simultaneous quantification of leprosy bacilli in clinical samples.     

An alternative, sensitive method to detect Helicobacter pylori DNA in feces

Background: Despite the high sensitivity and specificity of PCR, detection of Helicobacter pylori DNA in feces is still challenging. Fecal samples contain inhibitory molecules that can prevent amplification of the target DNA. Even by using specific DNA extraction kits for stools, monitoring of infection by analyzing stool samples remains problematic and endorses the need for improved diagnostic methods. Materials and Methods: The newly proposed method uses selective hybridization of target DNA with biotin‐labeled probes, followed by DNA isolation with streptavidin‐coated magnetic beads. After three washing steps, the purified DNA can be amplified immediately using conventional or quantitative PCR. In order to test this technique on biological samples, Mongolian gerbils were infected with H. pylori ATCC 43504 and fecal samples were analyzed on days 1, 4, and 10 post infection. Results: A detection limit of one bacterial cell per 100 mg stool sample was established, but only after removal of the magnetic beads from the target DNA by heating. This resulted in a 10‐fold increase of sensitivity compared to a commercially available stool DNA extraction kit. Analysis of fecal samples from infected gerbils demonstrated the presence of H. pylori DNA on each time point, while the uninfected animal remained negative. Conclusions: The proposed technique allows detection of very low quantities of H. pylori DNA in biological samples. In laboratory animal models, detailed monitoring of infection and complete clearance of infection can be demonstrated thanks to the low detection limit.     

An improved electroelution method for separation of DNA from humic substances in marine sediment DNA extracts

We present a method for the rapid and simple extraction of DNA from marine sediments using electroelution. It effectively separates DNA from compounds, including humic substances, that interfere with subsequent DNA quantification and amplification. After extraction of the DNA from the sediment into an aqueous solution, the crude sample is encased in 2% agarose gel and exposed to an electrical current, which draws the DNA out of the gel into a centrifugal filter vial. After electroelution, the sample is centrifuged to remove contaminants ≤100 000 Da. Recovery of DNA using this method is quantitative and does not discriminate on the basis of size, as determined using DNA standards and DNA extracts from environmental samples. Amplification of DNA is considerably improved due to removal of PCR inhibitors. For Archaea, only these purified extracts yielded PCR products. This method allows for the use of relatively large volumes of sediment and is particularly useful for sediments containing low biomass such as deeply buried marine sediments. It works with both organic-rich and -poor sediment, as well as with sediment where calcium carbonate is abundant and sediment where it is limited; consequently, adjustment of protocols is unnecessary for samples with very different organic and mineral contents.