Chain Reaction-based Methods For Detection Research Articles

Comparative analysis of cultural isolation and PCR based assay for detection ofCampylobacter jejuniandCampylobacter coliin foods of animal origin

The present study was designed to determine the occurrence of C. jejuni and C. coli in chicken meat, chicken offal, chevon and milk samples from Bareilly, Uttar Pradesh (India) to elucidate the role of these products in transmission of Campylobacter to humans. The samples were examined using both cultural as well as polymerase chain reaction based detection method. Out of 280 samples examined, 29 (10.36%) samples were found positive by cultural method, where as 68 (24.29%) samples showed presence of Campylobacter species by polymerase chain reaction. All the samples depicted positive by culture were also found positive by PCR. The highest occurrence rate was observed in chicken meat (46%), followed by chicken offal (20%), chevon (18%) and milk (3.75%). The results depicted the superior efficacy of PCR for rapid screening of samples owing to its high rapidity, specificity and automation potential. The study suggested the potential of transmission of Campylobacter species to humans due to the consumption of raw or contaminated meat or milk.

A sensitive polymerase chain reaction-based method for detection and quantification of metastasis in human xenograft mouse models

Tumor cell dissemination to distant organs accounts for the majority of cancer related deaths. Analysis of the stepwise process of metastasis formation and progression might provide novel therapeutic strategies for the treatment of disseminated cancer. However, studies with both biological and therapeutic endpoints would require highly sensitive and specific methods for precise quantification of the metastatic tumor burden in vivo. We have developed a quantitative real-time PCR-based assay for the detection and quantification of human tumor cells disseminated in mouse organs. The method relies on the parallel amplification of unique, species-specific, conserved and non-transcribed sequences in the mouse and human genomes. We tested the method in xenograft models to assess the metastatic potential of various cancer cell lines, the impact of injection modality and cell type on organ distribution, and the early stages of metastasis implantation and progression. With this method, we observed clear quantitative differences among colon cancer cell lines in terms of metastasis formation in the lung, consistent with the different in vitro growth properties. The mode of cell implantation and cell intrinsic properties strongly affected the metastatic pattern of prostate and breast cancer cell lines in mouse organs. The qPCR assay accurately determined the malignant cell burden even at early stages of metastasis progression in the lung. We describe a very sensitive assay for the highly reproducible detection and accurate quantification of human metastatic cells in mouse tissues and demonstrate its broad applicability to various experimental settings.

The evolving epidemiology of invasive meningococcal disease: a two-year prospective, population-based study in children in the area of Athens

In response to an increase in the incidence in invasive meningococcal disease (IMD) due to Neisseria meningitidis, a system of hospital- and laboratory-based surveillance was used in a prospective epidemiological and clinical assessment of IMD in children 0–13 years of age hospitalized in the Athens area between 1 January 1999 and 31 December 2000. The annual incidence of laboratory-confirmed disease was 10.2/100 000. Serogroup B strains were predominant. There was a sharp decrease in serogroup C from 19% of cases in 1999 to 3% in 2000 ( P=0.013). Of note was the emergence of serogroup A responsible for 7% of the cases. The overall case fatality rate was 4.5%, but 2.8% for microbiologically confirmed cases. A remarkable decrease in disease severity assessed by admissions to intensive care units was noted during the second study year. Polymerase chain reaction-based methods for detection of meningococcal DNA were the sole positive laboratory test in 45% of the cases and the only test on which serogroup determination was based in 52% of groupable cases. The epidemiological and clinical profile of meningococcal disease appears to be rapidly evolving and close monitoring is required particularly for input into decisions regarding use of meningococcal vaccines.

Polymerase Chain Reaction-Based Methods for Detection of Listeria monocytogenes: Toward Real-Time Screening for Food and Environmental Samples

A review is presented of nucleic acid amplification-based methodology, specifically polymerase chain reaction (PCR)-based assays, for the detection of Listeria monocytogenes in food and environmental samples. Until recently, developmental challenges including poor sensitivity, due in part to reaction inhibition by components of the sample matrix, and the potential for false-positive reactions have limited routine application of PCR-based screening assays. Commercial assays address these challenges while offering convenient, standardized protocols, a high level of automation, and results within 2 days after the sampling date. Although sample enrichment is necessary to achieve desired detection limits, continued efforts toward template purification will facilitate the development of assays offering real-time, quantitative results. The development of ribonucleic acid (RNA) amplification-based assays may increase in importance, particularly if end-product testing is prioritized by regulatory agencies, as messenger RNA appears to serve as an accurate indicator of cell viability. Further, the increase in target copy number may improve assay sensitivity. PCR-based screening methods offer efficient, reliable results and are ideal for monitoring the presence of L. monocytogenes in foods and in the food processing environment.

Chemotherapy of Chagas' disease: the how and the why.

Current developments in experimental chemotherapy of Chagas' disease are reviewed, in particular the demonstration that fourth-generation azole derivatives (inhibitors of sterol C14alpha demethylase), with particular selectivity against Trypanosoma cruzi and special pharmacokinetic properties, are capable of inducing radical parasitological cures in murine models of both acute and chronic disease. These are the first reports of parasitological cure of this disease in its chronic phase. We also discuss the relevance of etiological treatment in the clinical outcome of patients with chronic Chagas' disease. Although previous studies have suggested an important autoimmune component in the pathogenesis of this disease, recent results obtained using highly sensitive polymerase chain reaction based detection methods and detailed immunological characterization of the inflammatory process associated with chagasic cardiomyopathy indicate a positive correlation between tissue parasitism and the severity of cardiac pathological findings. Effective antiparasitic treatment can lead to regression of the inflammatory heart lesions and fibrosis in experimental animals and to stop the progression of the disease in humans. Taken together, these findings support the notion that the presence of the parasite is a necessary and sufficient condition for chagasic cardiomyopathy and confirm the importance of specific etiological treatment in the management of chronic chagasic patients.

A Polymerase Chain Reaction-Based Method for Detection and Quantification of Reporter Gene Expression in Transient Transfection Assays

Transcription in higher eukaryotes is often studied by the use of transient transfection assays. These experiments are performed by cloning putative cis-acting transcriptional elements (i.e., a promoter or enhancer) with a reporter gene that codes for a protein not expressed by the target cells. Although this approach is useful in many cases, the limited sensitivity of reporter assays can prevent studies in cases where few cells are obtainable or efficiency of transfection is low. We present an alternative approach. Cells are transfected with a plasmid containing a promoter with a human growth hormone (hGH) reporter gene. After an incubation period, RNA is isolated, and DNA complementary to the growth hormone mRNA is produced. The reporter cDNA concentration is measured by quantitative polymerase chain reaction (PCR). We have designed PCR primers that span the mRNA splice sites of the human growth hormone gene; these ensure exclusive amplification of the hGH cDNA and not the reporter plasmid. The assay is sensitive and simple to perform, requires no special equipment, and can quantify reporter cDNA concentration over a broad range.