Integrated Cell culture-PCR Research Articles

Detection of adenoviruses in shellfish by means of conventional-PCR, nested-PCR, and integrated cell culture PCR (ICC/PCR)

We tested three PCR based methodologies to detect adenoviruses associated with cultivated oysters. Conventional-PCR, nested-PCR, and integrated cell culture-PCR (ICC/PCR) were first optimized using oysters seeded with know amounts of Adenovirus serotype 5 (Ad5). The maximum sensitivity for Ad5 detection was determined for each method, and then used to detect natural adenovirus contamination in oysters from three aquiculture farms in Florianopolis, Santa Catarina State, Brazil, over a period of 6 months. The results showed that the nested-PCR was more sensitive (limit of detection: 1.2 PFU/g of tissue) than conventional-PCR and ICC-PCR (limit of detection for both: 1.2 x 10(2)PFU/g of tissue) for detection of Ad5 in oyster extracts. Nested-PCR was able to detect 90% of Ad5 contamination in harvested oyster samples, while conventional-PCR was unable to detect Ad5 in any of the samples. The present work suggests that detection of human adenoviruses can be used as a tool to monitor the presence of human viruses in marine environments where shellfish grow, and that nested-PCR is the method of choice.

Integrated cell culture/PCR for detection of enteric viruses in environmental samples.

Recently, an integrated cell culture/polymerase chain reaction (ICC/PCR) technique has been developed for the detection of viruses in environmental samples providing a reliable method for practical analysis and direct monitoring of environmental samples for viral pathogens. CC/PCR allows for detection of infectious viruses in hours to days compared with the days or weeks necessary with cell culture alone. Bacterial indicator organisms are commonly used to evaluate environmental samples with respect to fecal contamination and potential public health impacts. These organisms do not correlate well with the presence of viruses, but a rapid, reliable method was not previously available for direct virus testing. Using ICC/PCR, environmental samples may be directly surveyed for pathogenic viruses, in a timely manner. Direct virus analysis will lead to better assessment of the presence and risk of human enteric viruses in the environment, so that control measures may be developed with true virus occurrence data. The ICC/PCR approach combines two previously applied virus detection methods, conventional cell culture and PCR amplification, utilizing the major advantages and overcoming the major limitations of each methodology when used alone. Cell culture assay is the standard method for the detection of viable human viruses (i.e., poliovirus, coxsackievirus, echovirus, adenovirus, hepatitis A virus, reovirus, and rotavirus) in environmental samples, serving as the method against which all newer technologies are evaluated. Although cell culture is theoretically capable of detecting a single viable virus in relatively large volumes of sample, the time required for confirmed results with conventional cell culture makes it an impractical method for routine monitoring of environmental samples. Furthermore, cell culture does not detect noncytopathogenic viruses (viruses that are viable, infecting cells, and continually spreading to neighboring cells but that do not cause a visible cytopathogenic effect [CPE] on the cell monolayer). Rotavirus and most wild-type hepatitis A viruses (HAV) are infectious to cell cultures but do not produce a clear CPE.

MSP005 Genotyping of Candida tropicalis by different amplified fragment length polymorphism (AFLP) methods

This chapter discusses the theories and applications of various nucleic acid-based approaches such as gene probes, microarrays, conventional and real-time polymerase chain reaction (PCR), whole genome amplification, sequencing, comparative genomics, and metagenomics. Gene probes are single-stranded DNA, and can be used to identify a particular nucleic acid sequence within an environmental sample through colony hybridization, Southern and Northern hybridization (used for DNA and RNA respectively), Fluorescent in situ hybridization (FISH), microarrays, and phyloarrays. PCR is extremely sensitive and is able to detect a single target molecule in some cases. The use of PCR based techniques—such as reverse transcriptase–PCR (RT–PCR), introduced integrated cell culture-PCR (ICC-PCR), arbitrarily primed-PCR (AP-PCR), and Real-Time PCR has enabled the researchers to detect specific nucleic acid sequences in isolates as well in environmental samples. Recombinant DNA technology or DNA cloning has been widely used to examine the genetics of bacterial populations. Metagenomics is another nucleic acid-based approach that allows analysis of multiple genes and screening of large environmental fragments for functional activities. Several computer software sequence analysis programs and tools—such as Basic Local Alignment Sequence Tool (BLAST) or Clustal W—are available for sequences searches, and comparative genomics in such cases allows the comparison of subsets of genes or whole genomes of interest. Restriction fragment length polymorphism (RFLP) and its modifications such as amplified fragment length polymorphisms (AFLP) and terminal-restriction fragment length polymorphism (T-RFLP) can characterize or differentiate the whole genomes or specific PCR products from bacterial isolates. Denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electrophoresis (TGGE) has been employed to assess the microbial diversity. Plasmid analysis has been used to detect specific bacteria or genes such as in Bacillus anthracis.

Use of integrated cell culture-PCR to evaluate the effectiveness of poliovirus inactivation by chlorine.

Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.

Detection of infectious Cryptosporidium parvum oocysts in surface and filter backwash water samples by immunomagnetic separation and integrated cell culture-PCR.

A new strategy for the detection of infectious Cryptosporidium parvum oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable C. parvum to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious C. parvum in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the C. parvum KSU-1 hsp70 gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70 fragments, suggesting that several different strains of infectious C. parvum were detected.