Application of PCR for detection and identification of mycoplasma contamination in virus stocks

Summary A nested polymerase chain reaction (PCR) was used to detect and identify mycoplasma contaminants in viral stocks. The results of the PCR assay proved to be a sensitive and accurate indicator of the true status of the stock tested. Those samples positive by agar culture or Hoechst stain were also positive by PCR. Those samples that were inconclusive by Hoechst stain (10.05%) could be clearly determined to be mycoplasma positive or negative by PCR. The PCR assay also detected those fastidious species of mycoplasma that gave false negative results by the direct culture method. In many respects the PCR-based mycoplasma detection method described is superior to the agar culture and Hoechst staining detection methods. In this study, the PCR assay detected substantially more mycoplasma-positive viral stocks than did the agar culture assay. Due to its speed, sensitivity, and reliability, the PCR assay is of particular value in monitoring the process of removing mycoplasma from contaminated stocks. Furthermore, the PCR amplification products can be analyzed by restriction analysis to rapidly identify the species of the mycoplasma contaminating the stock tested.     

Evaluation of PCR as a means of identification of Pasteurella pneumotropica.

A polymerase chain reaction with new primers (new PCR) designed from Pasteurella pneumotropica 16S rDNA as an identification system for this organism was compared with the PCR reported by Wang et al. (Wang's PCR) by using 15 bacterial reference species and 70 clinical isolates with the conventional identification system. For the 15 reference strains, both PCRs were identical. For the 70 clinical isolates, the new PCR and Wang's PCR showed consistency with the conventional system in 62.9% (44/70) and 51.4% (36/70), respectively. Twenty-six isolates were inconsistent with the conventional system and the new PCR with respect to morphology and serology. These findings suggested that the new PCR was more sensitive than Wang's PCR, and the new PCR in combination with morphology and serology is useful for P. pneumotropica identification.     

Identification of adeno-associated virus contamination in cell and virus stocks by PCR

To further understand the biology of adeno-associated virus (AAV) and identify the presence of AAV in laboratory samples, we have developed a sensitive PCR-based assay using degenerate primers based on the sequence of seven diverse AAV isolates. Using these primers, we can detect free virus in viral stocks, cleared cell lysate, as well as in latently infected cells. The method can detect as little as 10 viral copies/microL of sample and can be adapted for high-throughput screening technology. With this method, we have also detected a new AAV isolate from a stock of bovine adenovirus.     

PCR detection of nearly any dengue virus strain using a highly sensitive primer 'cocktail'

PCR detection of viral pathogens is extremely useful, but suffers from the challenge of detecting the many variant strains of a given virus that arise over time. Here, we report the computational derivation and initial experimental testing of a combination of 10 PCR primers to be used in a single high-sensitivity mixed PCR reaction for the detection of dengue virus. Primer sequences were computed such that their probability of mispriming with human DNA is extremely low. A 'cocktail' of 10 primers was shown experimentally to be able to detect cDNA clones representing the four serotypes and dengue virus RNA spiked into total human whole blood RNA. Computationally, the primers are predicted to detect 95% of the 1688 dengue strains analyzed (with perfect primer match). Allowing up to one mismatch and one insertion per primer, the primer set detects 99% of strains. Primer sets from three previous studies have been compared with the present set of primers and their relative sensitivity for dengue virus is discussed. These results provide the formulation and demonstration of a mixed primer PCR reagent that may enable the detection of nearly any dengue strain irrespective of serotype, in a single PCR reaction, and illustrate an approach to the broad problem of detecting highly mutable RNA viruses.     

Rapid detection of low‐level HeLa cell contamination in cell culture using nested PCR

HeLa cells are a commonly used cell line in many biological research areas. They are not picky for culture medium and proliferate rapidly. HeLa cells are a notorious source of cell cross‐contamination and have been found to be able to contaminate a wide range of cell lines in cell culture. In this study, we reported a simple and efficient method for detecting the presence of HeLa cell contamination in cell culture. HPV‐18 was used as a biomarker. The cell culture supernatant was used directly as the template for nested PCR without extracting nucleic acid. By PCR amplification of the cell culture supernatant with the designed primers, we were able to detect the presence of HeLa cells in the culture. The sensitivity of this method can reach 1%, which is 10‐fold higher than Short tandem repeat sequence (STR) profiling. This simple, rapid, and “noninvasive” quality checking method should find applications in routine cell culture practice.     

Mink lung cells as a tool for detection ofMycoplasma hyorhinis contamination in cell cultures and virus stocks

Summary In an extensive host range study ofM. hyorhinis mink lung cells (MvlLu, ATCC CCL 64) were found to be the cells of choice for the propagation of this mycoplasm, which otherwise is often difficult to grow in a cell-free medium. Furthermore, rapid plaque assay and plaque purification procedures were developed forM. hyorhinis. The titer ofM. hyorhinis grew to 1×10⁷ to 1×10⁸ pfu/ml within three d postinoculation on mink lung cells. DNA restriction enzyme analysis of the genome ofM. hyorhinis was performed. Endonucleases Bst EII and Xho I are the most suitable enzymes for cleavingM. hyorhinis DNA into distinct fragment patterns. Thus, the use of the combination mink lung cells for mycoplasma growth with subsequent restriction enzyme analysis leads to an unamibiguous detection and identification toM. hyorhinis strains even in minute amounts.     

Hydroxyapatite adherence as a means to concentrate bacteria.

Adherence to hydroxyapatite (HA) was examined as a method to concentrate bacteria from foods. Using HA at a level of 10% and suspensions of an Escherichia coli strain containing 10(9), 10(6), and 10(3) cells per ml, kinetic studies revealed that maximum adherence was attained within 5 min for all cell concentrations and that comparable log reductions (1.0 to 1.5) of cells in suspension were seen regardless of initial cell concentration. Eleven species of spoilage and pathogenic bacteria were found to adhere to HA, with seven species adhering at proportions of greater than 95%. Fluorescent viability staining revealed that cells bound to HA remained viable. There was greater than 92% adherence of indigenous bacteria to HA from three of five 1:10 dilutions of ground beef, indicating promise for the use of HA for concentrating bacteria from meat and other food samples.     

Directed termination PCR: a one-step approach to mutation detection.

We describe a novel PCR-based method that allows the generation of nested termination fragments by integrating both selective DNA amplification and directed chain termination into a single PCR reaction. These termination fragments can be examined for sequence variation in either denaturing or non-denaturing polyacrylamide gels. This method provides a one-step and highly effective approach for the detection of both insertions/deletions and single base pair substitutions in sequences up to 1 kb in length.     

Accurate and efficient data processing for quantitative real-time PCR using a tripartite plant virus as a model

Real-time PCR is becoming a preferred method for quantification of minute amounts of nucleic acids. To achieve the full potential of this technique, accurate and convenient models for post-PCR data analysis are required. In this study, three different models were chosen to quantify the definitive copy numbers of Cucumber mosaic virus (CMV) genomic RNAs using raw fluorescence data of real-time PCR, and equations were proposed to compare their expression levels in virions or in planta. The results, as confirmed by standard curve and Northern blotting methods, show that the expression levels of different genes can be compared more accurately and more efficiently by these equations, especially using theoretical fluorescence (F0) and calibration factors (CF), determined by linear regression PCR (LinRegPCR). Thus, these equations, combined with data analysis by the LinRegPCR method, can greatly enhance the high-throughput quantification ability of real-time PCR, and permit accurate, reliable, and facile investigation of the changes in CMV RNAs accumulation.     

Lymphangiogenesis quantification using quantitative PCR and breast cancer as a model.

The detection of lymphangiogenesis (formation of new lymphatics) has previously been difficult to measure, primarily due to the lack of specific markers for lymphatic endothelium. Using conventional PCR (polymerase chain reaction), DNA sequencing, plasmid synthesis, and real-time quantitative PCR (RTQPCR), we report a new approach to enable the measurement of lymphangiogenesis using LYVE-1, a novel, specific lymphatic marker in breast cancer tissue. By using a Scorpion-based probe system with the RTQPCR analyser, a highly sensitive and specific detection and quantitation of LYVE-1 was possible. It was found that lymphangiogenesis occurred in all breast specimens and that higher levels were found in tumours which had spread to the lymph nodes.     

Application of PCR to human gene detection.

The advent of the polymerase chain reaction has stimulated the development of a number of rapid methods for characterizing human genes. In addition, the unprecedented level of sensitivity offered by some of these methods may prove to be of great value in the detection of minority cell populations. Over the past year, technical developments have been made in this area.     

Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR.

The suitability of a 16S rRNA-based mycoplasma group-specific PCR for the detection of mycoplasma contamination in cell cultures was investigated. A total of 104 cell cultures were tested by using microbiological culture, DNA fluorochrome staining, DNA-rRNA hybridization, and PCR techniques. A comparison of the results obtained with these techniques revealed agreement for 95 cell cultures. Discrepant results, which were interpreted as false negative or false positive on the basis of a comparison with the results obtained with other methods, were observed with nine cell cultures. The microbiological culture technique produced false-negative results for four cell cultures. The hybridization technique produced false-negative results for two cell cultures, and for one of these cell cultures the DNA staining technique also produced a false-negative result. The PCR may have produced false-positive results for one cell culture. Ambiguous results were obtained with the remaining two cell cultures. Furthermore, the presence of contaminating bacteria interfered with the interpretation of the DNA staining results for 16 cell cultures. For the same reason the hybridization signals of nine cell cultures could not be interpreted. Our results demonstrate the drawbacks of each of the detection methods and the suitability of the PCR for the detection of mycoplasmas in cell cultures.     

Campylobacter contamination in French chicken production from farm to consumers. Use of a PCR assay for detection and identification of Campylobacter jejuni and Camp. coli

AIMS: Campylobacter contamination in French chicken production from the farm to the consumer was determined using a PCR assay for bacteria detection and identification. METHODS AND RESULTS: Samples were bird droppings from poultry houses, neck skins, livers, hearts, gizzards, wings, legs and escalopes from slaughterhouses and gizzards, legs, drumstick, breast and escalopes from a supermarket. Bacterial DNA extraction was performed after an enrichment step in a broth and was followed by PCR. An internal control (IC) was used for both DNA extraction and PCR. Campylobacter were detected in 79.2% of poultry houses. Of the 303 samples, 201 were Campylobacter-positive (i.e. 66.3%) including 43.2% faecal samples, 5.6% slaughterhouse samples and 17.5% supermarket samples. There was no significant difference between the molecular method and the conventional culture technique for Campylobacter detection whatever the samples. The sensitivity was 5 UFC g(-1) of samples and 1.5 x 10(3) UFC ml(-1) of enrichment broth. The use of IC revealed PCR inhibition in 13 samples and problems in the DNA extraction in five samples. CONCLUSION: Significant Campylobacter contamination affects all stages of French chicken production. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of Campylobacter contamination at different levels of chicken production and the determination of the best place(s) for intervention are important for significantly decreasing Campylobacteriosis. Our technique is rapid and can be used on different chicken samples for Campylobacter detection and identification.