Development of polymerase chain reaction-based assays for bacterial gene detection

Polymerase chain reaction-based assays provide rapid, simple, and sensitive detection of bacterial genes, but are not without their drawbacks. This review summarizes the principal advantages and disadvantages of PCR-based bacterial gene detection, provides guidelines for the development and validation of new PCR assays, and describes potential pitfalls that may be encountered and how these can be avoided.     

Evaluation of four template preparation methods for polymerase chain reaction-based detection of Salmonella in ground beef and chicken

AIMS: To compare procedures for recovering template DNA from ground beef or chicken for polymerase chain reaction (PCR)-based detection of Salmonella. METHODS AND RESULTS: The primer set of ST11 and ST15 was utilized to amplify a 429-bp product from Salmonella serotype Typhimurium. Boiling and three commercial kits were evaluated for extracting DNA from pure suspensions and artificially contaminated ground beef and chicken. The detection sensitivity of the PCR assay for pure cultures was independent of the template preparation method (P=0.946). Boiling and GeneReleaser failed to detect Salm. Typhimurium at 4 x 106 cfu g(-1) in ground chicken. PrepMan Ultra and the high pure PCR template preparation kit facilitated reliable and sensitive detection of Salm. Typhimurium in two types of food. The sensitivities were approx. 4 x 103 cfu g(-1). When spiked samples were enriched in peptone water for 6 h, an initial inoculum of 1 cfu g(-1) was detectable. CONCLUSIONS: Four template DNA preparation methods differed in performance with respect to the type of samples tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Template DNA for the PCR detection of pathogenic bacteria, such as Salmonella in meat and poultry, could be effectively obtained using a simple rapid method such as the commercially available PrepMan Ultra kit.     

DNA polymerase chain reaction using fine needle aspiration biopsy smears to evaluate non-Hodgkin's lymphoma.

OBJECTIVE: To apply polymerase chain reaction (PCR) analysis to the fine needle aspiration biopsy (FNAB) evaluation of lymphoid proliferations. STUDY DESIGN: We analyzed 37 consecutive archived FNAB malignant lymphoma specimens. Immunophenotypic data from the fine needle aspiration biopsy and excisional biopsy material was available for all specimens. PCR to identify monoclonal rearrangements of the immunoglobulin heavy chain gene, T-cell receptor and translocations involving the bcl-1 and bcl-2 genes was performed. RESULTS: Seventy-eight percent of cases were detected by at least one of these assays. Where DNA analysis was performed on excisional biopsy material, 70% of the cases had identical results; no discordant results for the immunoglobulin heavy chain gene or T-cell receptor were found. In 23% of cases, after review of all available data, a discordant result was thought to be a consequence of a false negative result in DNA analysis of excisional biopsy material. CONCLUSION: These findings indicate that PCR analysis of archived FNAB material, when necessary, provides useful information for diagnosis and staging of malignant non-Hodgkin's lymphomas.     

A polymerase chain reaction-based method for isolation of gene-specific sequences from the interferon-alpha gene cluster.

The interferon-alpha gene is a gene family of over 20 distinct genes having 80-95% homology with one another at a nucleotide level. Because of the high homology in the gene cluster, the available interferon-alpha gene probes can hybridize to multiple bands of different size on Southern blot analysis of restricted human genomic DNA. We used the polymerase chain reaction with the primers synthesized from Alu repetitive sequence and the conserved sequences of the interferon-alpha gene cluster to generate specific probes for individual interferon-alpha genes. The amplification products were subcloned into a plasmid vector and analyzed by DNA sequencing and Southern blotting of the restricted human placental DNA. One clone, which derived from interferon-alpha 14 gene, produced a single 5.2-kb band in Southern blots of the HindIII-restricted human placental DNA. This stands in contrast to the 10 bands of different size that were detected with a cDNA for the interferon-alpha I' gene. Our results indicate that a polymerase chain reaction-based method can be used to isolate gene-specific sequences from the interferon-alpha gene cluster. Since a variety of human cancers has been found to have the complete or partial deletion of the interferon-alpha gene cluster, the gene-specific probe generated by this method may aid in determining the breakpoints in the vicinity of the gene cluster.     

Use of aqueous two-phase systems in sample preparation for polymerase chain reaction-based detection of microorganisms

An aqueous two-phase system, consisting of poly(ethylene glycol) (PEG) and dextran, was employed to separate polymerase chain reaction (PCR)-inhibitory substances from bacterial cells. The PCR inhibition of four soft cheeses was examined and three of them were found to be strongly PCR-inhibitory. Extraction of the PCR-inhibitory soft cheeses inoculated with Listeria monocytogenes in an aqueous two-phase system containing 8% (w/w) PEG 400 and 8% (w/w) dextran 500, was found to lower the PCR detection level of L. monocytogenes by more than four orders of magnitude in two of the cheeses compared to the case where no such sample pretreatment was performed. Depending on the type of cheese used, the PCR-inhibitory factors were found to be enriched in either the top or botton phase in the aqueous two-phase system. These results show that different soft cheeses contain different types and amounts of PCR-inhibitory substances.     

A hybrid-specific, polymerase chain reaction-based amplification approach for chromosomal walking

Here we report the development of an accurate and efficient genome walking approach based on ligation-mediated polymerase chain reaction (PCR) and magnetic capture hybrid selection technique. This approach overcomes the nonspecific amplification products that often occur in similar PCR-based methods. Our strategy was successfully applied for the cloning of the promoter region of the Cc RNase gene. This rapid, cost-effective, and sensitive protocol can easily be extended for use in the isolation and amplification of any target sequences for which only partial information is known such as identification of the position of transposable elements and integrated viral DNA sequences.     

Construction of tandem repeats of DNA fragments by a polymerase chain reaction-based method

We describe a new application of megaprimer polymerase chain reaction (PCR) for constructing a tandemly repeated DNA sequence using the drought responsive element (DRE) from Arabidopsis thaliana as an example. The key feature in the procedure was PCR primers with partial complementarity but differing melting temperatures (T(m)). The reverse primer had a higher T(m), a 3' end complementary to the DRE sequence and a 5' region complementary to the forward primer. The initial cycles of the PCR were conducted at a lower primer annealing temperature to generate products that served as megaprimers in the later cycles conducted at a higher temperature to prevent annealing of the forward primer. The region of overlap between the megaprimers was extended for generating products with a variable copy number (one to four copies) of tandem DRE sequence repeats (71?bp). The PCR product with four tandem repeats (4× DRE) was used as a template to generate tandem repeats with higher copies (copy number large than four) or demonstrated to bind DRE-binding protein in an yeast one-hybrid assay using promotorless reporter genes (HIS and lacZ). This PCR protocol has numerous applications for generating DNA fragments of repeated sequences.     

Rapid preparation of DNA from phytopathogenic mycoplasma-like organisms for polymerase chain reaction analysis

A simple procedure for the processing of MLO (mycoplasma-like organisms)-infected plant samples for PCR analysis is described. It is based on differential centrifugation and removal of enzyme inhibiting agents with the aid of a resin. Results of the amplifications are comparable with those achieved with more complex DNA extraction procedures. The method does not require any extraction with organic solvents or alcoholic precipitation of nucleic acids.     

A polymerase chain reaction-based method to detect cisplatin adducts in specific genes.

Every bulky lesion in DNA can potentially inhibit the Taq DNA polymerase and thereby decrease the amplification produced in the polymerase chain reaction. We investigated the feasibility of using this inhibition to quantify DNA lesions produced by the anticancer drug cisplatin. Products were detected by electrophoresis followed by ethidium bromide staining. Quantitation was obtained by including [32P]dCTP in the amplification reaction and subsequently assessing the incorporated radioactivity. Hamster genomic DNA was platinated in vitro to defined levels and amplified with primers that produce either a 150, 750 or 2,000 base pair fragment. The degree of inhibition of PCR agreed with the predicted level of DNA platination in each size of fragment, suggesting that the polymerase was inhibited by every cisplatin-induced lesion. This method was used to detect cisplatin-induced lesions in the adenine phosphoribosyltransferase gene of CHO cells. Cells were incubated with 0-125 microM cisplatin for 2 h, the DNA was purified and subjected to PCR. A significant decrease in amplification of the 2 kbp fragment was observed in DNA from cells incubated with cisplatin at 75 microM. The degree of inhibition agreed closely with the amount of DNA damage in the overall genome as measured by atomic absorption. No change was detected in amplification of the 150 base fragment which can therefore be used to normalize data for any variations between DNA samples. This assay has the same sensitivity as other methods currently used for the analysis of gene-specific damage. The advantage of this assay is that it obviates the need for specific endonuclease complexes to recognize and cleave DNA adducts as previously required when analyzing damage in specific genomic sequences.     

Campylobacter jejuni: Specific oligonucleotides and DNA probes for use in polymerase chain reaction-based diagnosis

Abstract A 1189 base-pair long DNA fragment, VS1, was isolated from a Campylobacter jejuni CIP 70.2 cosmid library and was found to contain regions specific for this bacterial species. For detection and identification of C. jejuni , two oligonucleotides derived from the VS1 sequence were used as primers in polymerase chain reaction test on genomic DNAs from 38 Campylobacter and from 10 non- Campylobacter strains. A specific, 358 base-pair long DNA fragment was amplified only when C. jejuni DNA was used as a target. The detection limit of the amplification reaction was as low as 1.86 fg DNA, which is the equivalent of one C. jejuni genome.     

Negative selection of thymocytes. A novel polymerase chain reaction-based molecular analysis detects requirements for macromolecular synthesis.

Self-tolerance is mainly established through clonal deletion of autoreactive T cells during thymic differentiation. The mechanisms by which deletion is achieved are poorly understood. Here we use a specific polymerase chain reaction-based system to characterize DNA fragmentation and show that after in vivo treatment of neonatal mice with staphylococcus enterotoxin B, selective apoptosis of V beta 8+ thymocytes occurs. This process precedes detectable deletion of V beta 8+ cells as determined by phenotypic analysis. Moreover, in vivo administration of cycloheximide and, to a lesser extent, actinomycin D, inhibits apoptosis of staphylococcus enterotoxin B specific thymocytes. Thus, macromolecular synthesis is a requirement for negative selection.     

A comparison of methods for DNA extraction from paraffin-embedded tissue for microsatellite instability analysis by polymerase chain reaction

In a prospective study, nuclear DNA was extracted from colorectal tumours and normal mucosa which had been fixed in buffered formalin and embedded into paraffin. DNA-extraction was performed using three different methods: a commercial kit which was not especially created for this use; a known fast procedure without DNA-cleaning steps; and a more conventional DNA-preparation protocol with DNA-cleaning. Using the polymerase chain reaction (PCR), DNA was amplified by being targeted onto two β-globin fragments with different lengths (536 bp and 989 bp) and (CA)n repeats localized on chromosome 5q (D5S346) and chromosome 17p (TP53CA) with a length of about 100 bp for detection of microsatellite instability. The success rate of microsatellite amplification was 100% with all methods. The 536 bp β-globin fragment could be amplified with a success rate ranging from 40% to 100%. The amplification of the 989 bp β-globin fragment was unsuccessful. Significant differences were observed between the three methods in the final DNA concentration and DNA yield. In microsatellite instability studies of paraffin-embedded tissues, the investigator can expect a high success rate of nearly 100% using any of the described methods.     

A simple polymerase chain reaction-based method for the construction of recombinase-mediated cassette exchange donor vectors

Here we describe a simple method for generating donor vectors suitable for targeted transgenesis via recombinase-mediated cassette exchange (RMCE) using the ΦC31 integrase. This PCR-based strategy employs small attB “tails” on the primers used to amplify a sequence of interest, permitting the rapid creation of transgenes for in vivo analysis.     

Optimal conditions and specific characteristics of Vent exo- DNA polymerase in ligation-mediated polymerase chain reaction protocols.

An optimized procedure for the ligation-mediated polymerase chain reaction (PCR) technique using Thermococcus litoralis exo- DNA polymerase (Vent exo-) was developed. The optimal dosage of Vent exo- at the primer extension and PCR amplification steps as well as the optimal DNA quantity to use were established. We showed that Vent exo- can efficiently create the blunt-ended termini required for subsequent linker ligation. Vent exo- proves to be more efficient than Pyrococcus furiosus exo- (Pfu exo-) for this task. Vent exo- resolves highly GC-rich sequence substantially better than Thermus aquaticus DNA polymerase (Taq) and with a similar efficiency as Pfu exo-. The DNA/DNA polymerase activity ratio is significantly higher for Vent exo- than for Pfu exo-, which is reflected by the sensibility of Vent exo- in efficiently amplifying genomic DNA. Furthermore, the range of efficiency of Vent exo- demonstrates the importance of conducting evaluative testing to identify the optimal dosage of use of this polymerase to obtain successful PCR amplification. Optimal MgSO4 concentrations to use with Vent exo- were established. Our results show that Vent exo- DNA polymerase produces bands of uniform and strong intensity and can efficiently be used for the analysis of DNA in living cells by ligation-mediated PCR.     

A real-time polymerase chain reaction-based method for rapid and specific detection of spoilage Alicyclobacillus spp. in apple juice

AIMS: To develop a real-time PCR-based rapid detection method for spoilage Alicyclobacillus spp. in juice products. METHODS AND RESULTS: The squalene-hopene cyclase-encoding gene was targeted for primer-and-probe development. Gene fragments from representative strains were cloned, and PCR primers and probe were designed by DNA sequence comparison. Selected bacteria were examined for cross-reactivity by the new method. Cells were serially diluted in apple juice and saline, and examined by the new method to establish detection sensitivity. Using the newly developed Taqman real-time PCR-based method, strains of Alicyclobacillus acidocaldarius and A. acidoterrestris were detected without cross reactivity with other common food-borne micro-organisms. Detection of <10 cells per PCR reaction from juice samples was accomplished within 3-5 h. CONCLUSION: This is the first reported real-time PCR-based detection method for Alicyclobacillus spp. and its application in juice products is demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: As a favourable alternative for the laborious and time-consuming culture- or biochemical characterization-based techniques, the system has great potential for industrial applications from raw material screening to final product quality control.     

Clinical performance of non-radioactive assays for HIV-1 DNA amplified by the polymerase chain reaction

A major factor preventing more widespread use of polymerase chain reaction in the clinical laboratory is the lack of convenient non-radioactive probe hybridization procedures which do not sacrifice sensitivity or specificity. In this report, we describe comparisons of probes labelled with biotin, digoxygenin, alkaline phosphatase, and³²P. We report the comparison of solution or liquid hybridization assay and Southern blotting with digoxygenin-labelled oligonucleotides on a total of 64 clinical specimens. Perfect diagnostic agreement between the³²P and digoxygenin probes was obtained. These data suggest that the non-radioactive assay as described is as sensitive and as specific as the assay with³²P-Iabelled probes.     

Genotypic analysis of DNA isolated from fine needle aspiration biopsies

DNA was isolated from 20 fine needle aspiration (FNA) biopsies from lymphomas, hyperplastic lymph nodes and nonlymphoid malignant tumors. Small aliquots (0.2 microgram to 2.0 micrograms) of DNA from each sample were digested to completion with restriction endonuclease Eco RI and/or Bam HI and electrophoresed in 0.8% agarose minigels. DNA was transferred to a nylon filter after brief treatment in HCl and subsequent denaturation and neutralization. Filters were hybridized to radiolabeled JH, C kappa, TCR beta or bcl-2 probes to determine if these genes were in germline or rearranged configurations in each of the samples. It was possible to demonstrate rearrangement of at least one immunoglobulin gene in each of the samples diagnosed as lymphoma, while all samples derived from hyperplastic lymph nodes and nonlymphoid malignant tumors exhibited a germline pattern for each probe tested. Thus, FNA biopsies can provide suitable and sufficient DNA for genotypic analysis using molecular probes that detect gene rearrangement.     

Re-usable DNA template for the polymerase chain reaction.

DNA covalently bound to an uncharged nylon membrane was used for consecutive amplifications of several different genes by PCR. Successful PCR amplifications were obtained for membrane-bound genomic and plasmid DNA. Membrane-bound genomic DNA templates were re-used at least 15 times for PCR with specific amplification of the desired gene each time. PCR amplifications of specific sequences of p53, p16, CYP1A1, CYP2D6, GSTM1 and GSTM3 were performed independently on the same strips of uncharged nylon membrane containing genomic DNA. PCR products were subjected to restriction fragment length polymorphism analysis, single-strand conformational polymorphism analysis and/or dideoxy sequencing to confirm PCR-amplified gene sequences. We found that PCR fragments obtained by amplification from bound genomic DNA as template were identical in sequence to those of PCR products obtained from free genomic DNA in solution. PCR was performed using as little as 5 ng genomic or 4 fg plasmid DNA bound to membrane. These results suggest that DNA covalently bound to membrane can be re-used for sample-specific PCR amplifications, providing a potentially unlimited source of DNA for PCR.