Single Primer Pair Research Articles

Applicazioni della PCR e PCR in situ nella diagnosi di infezioni batteriche e virali da biopsie fissate in formalina e incluse in paraffina

In situ PCR, amplification of target DNA sequences in fixed cells, is a very useful molecular biology tecnique with potential to combine the high sensitivity of tube PCR with the precise anatomical localization of the targeted bsequences. It allows the study of low copy viral or bacterial DNA. In this study we document the utility of directin situ PCR with single primer pair by applying it to 3 infectious agents in different model systems: Borrelia burgdorferi in 5 Eritema migrans lesions and 55 primitive cutaneous B cell lymphomas, Chlamydia pneumoniae in 200 autoptic atheromasic lesions, and Papilloma virus in 20 CIN 1 (mild cervical dysplasia). In situ PCR seems to be a very promising tecnique; however, the prerequisite for the success of in situ PCR is conditioned by optimal standardization of the key variables which, on the other hand, are influenced by tissue composition.

Detection and identification of Bartonella species pathogenic for humans by PCR amplification targeting the riboflavin synthase gene (ribC).

Several Bartonella species have now been implicated as human pathogens. The recovery of these fastidious organisms in the clinical microbiology laboratory remains difficult, and current methods are still relatively insensitive. Thus, the bartonellae are good candidates for detection by PCR. We have developed a PCR assay which uses a single primer pair targeting the riboflavin synthase gene (ribC) and detected six Bartonella species that have been implicated in human disease, B. henselae, B. quintana, B. bacilliformis, B. clarridgeiae, B. elizabethae, and B. vinsonii subsp. berkhoffii. Species identification is achieved simply by restriction enzyme digestion of the amplicon. This PCR assay appears to be specific for the Bartonella genus because it failed to amplify DNA from several other bacterial species.

Simple PCR-based method for synthesis of molecular calibrators and controls.

The use of molecular methods such as PCR to quantify nucleic acids has become widely established for both research and diagnostic applications. Increasingly, such methodologies recognize the need for inclusion of internal standards and controls. These are used to define the efficiencies of nucleic acid extraction, reverse transcription, amplification, and detection and to quantify the effects of nonspecific inhibitors. Examples include the “Qs” controls included in Amplicor PCR assays (Roche Molecular Systems Inc.) (1) and the “Qa, Qb, Qc” controls used in Nuclisens assays (Organon Technica) (2). Control constructs (RNA or DNA) are derived from the nucleic acid sequence that the test method is designed to assay and span the primer binding sequences used in the assay. Thus, when added to the test sample, the control is coamplified in the reaction with a single primer pair. To distinguish the control from the test sequence during the detection phase of the assay, a short sequence between the primer binding sites is replaced by a defined sequence to allow differential probing with labeled oligonucleotides. Nucleic acid controls are usually 1 kb or longer, as shorter lengths may not by efficiently purified by the commonly used silica capture or precipitation methods. Previously, the construction of such controls required the identification or engineering of unique restriction sites between the primer binding sites to allow excision of the wild-type sequence and replacement with the probe sequence (1)(2). The process also required ligation of the probe sequence into the target sequence backbone and cloning into a plasmid containing a transcription promoter in the case of RNA controls. The method described here simplifies the procedure to a two-stage PCR amplification and allows the probe sequence to be placed anywhere between the primer binding sites without the need to identify or engineer restriction sites. Similar approaches …

A single primer pair immunoglobulin polymerase chain reaction assay as a useful tool in fine-needle aspiration biopsy differential diagnosis of lymphoid malignancies.

Different polymerase chain reaction (PCR) assays have proved to be useful tools for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements in frozen or paraffin-embedded tissues. However, the usefulness of a single primer pair PCR assay on fine-needle biopsy aspirates has not been proven yet. The authors conducted a wide prospective study of 148 lymph nodes and extranodal lymphoid infiltrates obtained by fine-needle aspiration biopsy (FNAB). The power of a single primer pair PCR amplification of the hypervariable CDRIII region IgH genes was evaluated using a pair of consensus primers. The PCR cytologic results were compared with the final clinicopathologic diagnosis of each case assessed by combining cytologic and/or immunophenotypic data and histologic features or clinical follow-up. Among the 139 cases with an evaluable PCR result, 35 of 40 (87%) B-cell non-Hodgkin lymphomas were detected as a monoclonal band. Monoclonal IgH bands also were detected in two of the five (40%) T-cell lymphomas, two of the seven (29%) Hodgkin lymphomas, and 5 of the 87 (6%) reactive lymphoid disorders. These results are similar to those obtained by other authors using seminested PCR or combining different PCR tests in each sample obtained by FNAB or excisional biopsy. The results of the current study demonstrate the convenience of a single primer pair PCR amplification over seminested methods in terms of lower cost and workload. The existence of PCR false-negative and false-positive results for lymphoma makes it necessary to combine the information obtained by PCR with cytologic and/or immunophenotypic data to optimize the number of lymphoid malignancies correctly diagnosed by FNAB.

DETECTION OF N-MYC GENE AMPLIFICATION IN NEUROBLASTOMA BY COMPARATIVE, IN SITU,AND REAL-TIME POLYMERASE CHAIN REACTION

We have used semiquantitative comparative and real-time quantitative polymerase chain reactions (PCR) to detect n-myc gene-amplification in 20 frozen neuroblastoma biopsies and IMR 32 cell line to predict biological behavior of the tumors. Two primer pairs were used for the semiquantitative method to co-amplify a 520-bp fragment of the beta-globin gene--used as a single copy reference standard--and a 258-bp fragment of the n-myc gene. After 30 cycles the PCR products were electrophoresed through an agarose gel and were compared to each other with use of a gel-densitometer. Real-time quantitative analyses were performed in a LightCycler instrument. A single primer pair was used to amplify a 120-bp fragment of the n-myc oncogene and a LC640-labeled fluorescent probe pair to detect the product. Calibration curve, set up from a serial dilution including samples with 1, 2, 10, 13, 25-fold n-myc oncogene amplification, was used for quantitative analysis. The semiquantitative method did not show distinct difference between tumor groups with no amplification and less than 10-fold amplification, whereas quantitative LightCycler analysis was able to detect even 2-fold amplification. Differentiated neuroblastomas seldom show n-myc amplification. In spite of this, we have found two partly differentiated tumor samples that contained n-myc amplification. In these cases in situ PCRs were performed to examine the tumor heterogeneity. We used biotinated ATP labeling and the same primer pair as for the LightCycler analysis. In both cases differentiated cells did not show n-myc gene amplification, whereas considerable amplification was detected in the neuroblasts.

Development and transferability of apricot and grape EST microsatellite markers across taxa.

EST microsatellite markers were developed in apricot (Prunus armeniaca L.) and grape (Vitis vinifera L.). cDNA libraries from either apricot leaves or grape roots were used in an enrichment procedure for GA and CA repeats. The transferability of EST simple sequence repeat (SSR) markers from apricot and grapevine to other related and unrelated species was examined. Overall, grape primers amplified products in most of the Vitaceae accessions while the apricot primers amplified polymorphic alleles only in closely related species of the Rosaceae. In this taxonomic family, ten EST SSR loci were tested, and one single primer pair, PacB22, was amplified across species and sections in the Prunoideae and Maloideae. Sequencing of EST SSR loci in other species and genera confirmed a higher level of conservation in the microsatellite motif and flanking regions in the Vitaceae compared to the Rosaceae. Two distinct fragments of the PacB22 locus amplified across the Malus and Pyrus genera; however, while the coding region was highly conserved, the microsatellite repeat motif was no longer present. The banding pattern was explained by base substitution and insertion/deletion events in the intronic region of PacB22. This study includes the determination of the degree of polymorphism detected among species and genera in two unrelated taxonomic families and the evaluation of the information provided by the microsatellite repeats and the flanking regions.

PCR-RFLP using Ssu-rDNA amplification as an easy method for species-specific diagnosis of Trypanosoma species in cattle.

A single polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay was used to characterise all important bovine trypanosome species. This is the first report of a sensitive pan-trypanosome PCR assay amplifying all species including T. vivax to a comparable extent using a single primer pair. A semi-nested PCR approach resulted in the detection of one T. congolense trypanosome genome/40 microl of blood, applied as buffy coat on filter paper. Restriction enzyme analysis using Msp1 and Eco571 gave a clear distinction between T. congolense, T. brucei, T. vivax and T. theileri. Several subgroups within the T. congolense group could be distinguished but no differences between the species belonging to the subgenus Trypanozoon or between T. simiae and T. theileri could be found. The use of MboII restriction enzyme allowed differentiation between T. simiae and T. theileri. The potential of the essay to be used as a suitable diagnostic tool is discussed.

Detection and differentiation of serologically cross-reacting tobamoviruses of economical importance by RT-PCR and RT-PCR-RFLP

A procedure involving reverse transcription followed by the polymerase chain reaction (RT-PCR) using a single primer pair was developed for the detection of five tobamovirus species which are related serologically. Either with a subsequent restriction enzyme analysis (RT-PCR-RFLP) or with a RT-PCR using species specific primers the five species can be differentiated. To differentiate those species by serological means is time consuming and might give ambiguous results. With the example of the isolate OHIO V, which is known to break the resistance in a selection of Lycopersicon peruvianum, the suitability of the RT-PCR-RFLP technique to detect variability at the species level was shown. In sequence analysis 47 codons of the coat protein gene of this isolate were found to be mutated compared to a tobacco mosaic virus (TMV) coat protein gene sequence. Forty of these mutations were silent and did not change the amino acid sequence. Both procedures are suitable to detect mixed infections. In addition, the RT-PCR-RFLP give information on the relative amounts of the viruses that are present in a doubly infected plant. The RT-PCR-RFLP using general primers as well as the RT-PCR using species specific primers were proven to be useful for the diagnosis and control of the disease and will be helpful for resistance breeding, epidemiological investigations and plant virus collections.

Rapid Detection of Escherichia coli O157:H7 with Multiplex Real-Time PCR Assays

A SYBR Green LightCycler PCR assay using a single primer pair allowed simultaneous detection of stx1 and/or stx2 of Escherichia coli O157:H7. A distinct sequence of the Shiga-like toxin genes was amplified to yield products of 227 and/or 224 bp, respectively. The two products were distinguished by melting point curve analysis.

GC-rich template amplification by inverse PCR. DNA polymerase and solvent effects.

The amplification of GC-rich templates by any PCR method is usually a difficult task and despite the development of modified methods and conditions, this type of amplification still remains a specific case approach. Problems usually observed with GC-rich DNA are constraint of template amplification by stable secondary structures that stall or reduce the DNA polymerase progress, and the presence of secondary annealing sites giving rise to nonspecific amplified bands. This latter point is not exclusive to GC-rich templates but is frequently encountered in other types of templates. In order to design a more general method for GC-rich templates, different DNA polymerases were compared in combination with different organic solvents with the purpose of abolishing stable secondary structures (). Our attention focused on the inverse polymerase chain reaction (iPCR) used to perform site-directed mutagenesis ( ). This very attractive method req⪚res a single pair of primers and involves the amplification of the whole recombinant plasmid, a difficult step with high GC-content DNA. Inverse PCR also proves useful in cloning missing parts of genes by using a self-ligated genomic DNA fragment as template.

Simultaneous RT/PCR detection and differentiation of arabis mosaic and grapevine fanleaf nepoviruses in grapevines with a single pair of primers

The movement protein genes from several isolates of ArMV and GFLV of different geographical origins were amplified by RT/PCR using degenerate primers, cloned and sequenced. A single pair of degenerate primers was designed from these sequences to allow the simultaneous amplification of parts of the movement protein genes of ArMV and GFLV. Their use in an immunocapture-RT/PCR for the detection of ArMV or GFLV in infected grapevines proved to be ten times more sensitive than the corresponding ArMV or GFLV ELISA tests. A Sph1 restriction site found in the sequences corresponding to the amplified products from the GFLV isolates, but not in the amplified products from the ArMV isolates, allowed the differentiation between ArMV and GFLV in the infected grapevines by a Sph1 restriction digestion of the amplified products.

Development of a Brucella suis specific hybridisation probe and PCR which distinguishes B. suis from Brucella abortus

A genomic library was prepared from Brucella suis DNA ( MboI digested) and cloned into the BamHI site of pUC18. Colony hybridisation using a probe prepared from purified B. suis DNA labelled with α 32P was carried out to identify colonies of interest. About 20 colonies, which gave an intense signal upon hybridisation with whole B. suis genomic DNA as a probe, were selected. Because of the high degree of DNA homology between B. suis and Brucella abortus, a short probe was chosen as it would more likely give species specificity. Of seven fragments selected to probe whole B. suis, B. abortus, and Yersinia enterocolitica DNA, one was found to hybridise with B. suis only. The probe was sequenced in two directions and sense and anti sense primers of 25 bp in length were chosen to yield a product of 421 bp. After optimisation of the PCR, a product of 420 bp was obtained with B. suis template DNA and two bands of 420 and 650 bp were detected with B. abortus template DNA. This is the first reported PCR of the Brucella genome where a single pair of primers will discriminate between B. suis and B. abortus. No band was observed when the two primers were used to amplify E. coli, Y. enterocolitica, Enterobacter cloacae, Staphylococcus aureus, Streptococcus uberis, Corynebacterium bovis, or Serratia marcescens template DNA.

Amplified Fragment-Length Polymorphism, a New Method for the Analysis of Brewer's Yeast DNA Polymorphism

This study sought to confirm the utility of amplified fragment-length polymorphism for identifying brewery yeast strains. Our results were promising, since single primer pairs can be used to distinguish most yeast strains. The use of several primer pairs, however, should still increase the method's reproducibility. Furthermore, this technique yields quantitative data on genetic polymorphism. Among the 26 strains studied, we calculated a 55% average of shared fragments. This similarity indicator was higher for bottom-fermenting strains (72%) than for top-fermentation yeasts (45%).

AFLP Analysis of Opium Poppy

Amplified restriction fragment length polymorphic (AFLP) DNA analysis was performed on leaf samples of 40 accessions of opium poppy (Papaver somniferum L.) and two other control genera (Papaver bracteatum Lindley and Papaver setigerum DC.) from a commercial breeding collection held in Tasmania, Australia. A similarity dendrogram was produced on the basis of the analysis of all AFLP bands that ranged between 66 and 367 base pairs (bp) seen on an autoradiogram from denaturing polyacrylamide sequencing electrophoretic gels with three different primer pairs. It was necessary to combine the analysis of all three primer pairs into a single dendrogram to ensure that replicate analyses of related populations were grouped together. It was necessary to use more than one primer pair to resolve clearly the genetic relationship between the closely related plants described in this study; however, even a single primer pair could easily distinguish between populations of different poppy species. The application of AFLP DNA analysis for cultivar identification represents an efficient, reliable procedure for identifying opium poppy breeding lines in a definitive manner. This procedure required only a small amount of leaf material for analysis and the degree of the genetic relatedness was performed on autoradiographic banding patterns with commercially available software. These data were used to generate a similarity dendrogram depicting the predicted genetic relationship of the opium poppy cultivars. Opium poppy accessions sharing common parental lines could be distinguished unambiguously from poppy accessions of more distant genetic background. The banding patterns were reproducible, consistent within a genotype, and the DNA polymorphisms were frequent enough to be useful in characterizing genetic diversity in even closely related breeding lines.

Effect of primers hybridizing to different evolutionarily conserved regions of the small-subunit rRNA gene in PCR-based microbial community analyses and genetic profiling.

Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (alpha, beta, and gamma subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking "universal" primers can affect a PCR-based microbial community analysis.

Determination of the Relative Expression Levels of Rubisco Small Subunit Genes in Arabidopsis by Rapid Amplification of cDNA Ends

Multigene families are common in higher organisms. However, due to the close similarities between members, it is often difficult to assess the individual contribution of each gene to the overall expression of the family. In Arabidopsis thaliana, there are four genes encoding the small subunits (SSU) of ribulose-1.5-bisphosphate carboxylase oxygenase (rubisco) whose nucleotide sequences are up to 98.4% identical. In order to overcome the technical limitations associated with gene-specific probes (or primers) commonly used in existing methods, we developed a new gene expression assay based on the RACE (rapid amplification of cDNA ends) technique with a single pair of primers. With this RACE gene expression assay, we were able to determine the relative transcript levels between four Arabidopsis SSU genes. We found that the relative SSU gene expression differed significantly between plants grown at different temperatures. Our observation raises the possibility that an adaptation of rubisco to the environment may be achieved through the specific synthesis of the SSU proteins, which is determined by the relative expression levels between the SSU genes.

A single PCR-restriction endonuclease analysis for rapid identification of Malassezia species

The present study describes a system based on PCR and restriction endonuclease analysis (REA) to distinguish the seven currently recognized Malassezia species. Fifty-five representative yeast isolates were examined. A single primer pair was designed to amplify the large subunit ribosomal RNA (LSU rRNA) gene of the seven Malassezia species, and identification was achieved by digestion of the PCR products with three restriction endonucleases: BanI, HaeII and MspI. A specific restriction endonuclease analysis pattern was determined for each species investigated. Moreover, PCR-REA allowed the detection and characterization of mixtures of several Malassezia species. PCR-REA of only the LSU rRNA gene is a reliable and rapid method to distinguish all Malassezia species. PCR-REA represents a considerable saving in time over currently available identification procedures. This method should be evaluated on clinical material directly.

PCR-heteroduplex analysis of T-cell receptor gamma gene rearrangement in paraffin-embedded skin biopsies.

We developed a rapid, simple, and sensitive method for the detection of T-cell receptor-gamma (TCRgamma) gene rearrangements in paraffin-embedded skin biopsies. Available techniques often require either fresh tissue, several primer pairs, nested amplifications, or specialized electrophoresis steps such as denaturing gradient gel electrophoresis. Our method is based on heteroduplex analysis of polymerase chain reaction (PCR) products of the TCRgamma in a nondenaturing modified polyacrylamide gel using a single pair of primers and is adapted for paraffin-embedded tissue. When tested against Southern blot analysis, the PCR results correlated in 8 of 9 cases. Six mature cutaneous B-cell lymphomas and 29 inflammatory skin disorders all resulted in a polyclonal amplification pattern. When analyzing 3-mm or 4-mm punch biopsies of 51 cases of cutaneous T-cell lymphoma, 37 (72.5%) showed a clonal rearrangement with this technique. For 7 cases of patch stage mycosis fungoides, frozen tissue and formalin-fixed and paraffin-embedded tissue was available, and in 5 of 7 cases (71%), the results in frozen and paraffin-embedded tissue were concordant. One case showed a clonal pattern in frozen tissue but not in paraffin-embedded tissue, and one case was polyclonal in frozen tissue but monoclonal in paraffin-embedded tissue. Using serial dilutions of DNA from a T-cell ALL in a polyclonal background (tonsil), we established a sensitivity of 0.5%. Heteroduplex PCR of the TCRgamma is a rapid, sensitive, and inexpensive screening procedure as well as a useful adjunct to histologic analysis and immunophenotyping of cutaneous T-cell proliferations.

Simultaneous Quantitation of Topoisomerase II α and β Isoform mRNAs in Lung Tumor Cells and Normal and Malignant Lung Tissue

Certain drugs used in the treatment of lung cancer and other human malignancies are cytotoxic because of their ability to interact with the two isoforms of topoisomerase II (topo II), topo IIα and topo IIβ. As part of an effort to evaluate the contribution of topo II alterations to drug sensitivity and resistance in lung cancer, we have developed a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to measure levels of topo II α and β mRNAs simultaneously using a single pair of primers with sequences common to both isoforms. The PCR products derived from the topo II α and β mRNAs are both 446 bp but have different electrophoretic mobilities in a nondenaturing polyacrylamide gel, allowing sensitive, rapid quantitation when the products are radiolabeled with [35S]-dATP. Using this RT-PCR method, poly(A+) RNA from 13 non-small cell lung cancer (NSCLC) cell lines was analyzed. The results obtained indicated that the cell lines express a wide range of topo IIα mRNA levels (12-fold) and topo IIβ mRNA levels (5.5-fold). Tumor and normal lung tissues from 25 patients with NSCLC were also examined. In the tumor samples, the levels of the topo II α and β mRNAs were similar. However, mean topo IIα mRNA levels in the tumors were approximately 7-fold higher than those of the paired normal lung tissues. In contrast, topo IIβ mRNA levels were similar in both tumor and normal lung. Topo II α and β mRNA levels were both significantly lower in the squamous cell tumors than in the adenocarcinoma samples. Topo IIβ mRNA levels in the squamous cell tumors were also significantly lower than those in paired normal lung tissue. The RT-PCR method described is reliable and convenient, and for the first time, makes the rapid simultaneous direct comparison of topo IIα and topo IIβ mRNA levels feasible in large numbers of clinical samples.

Development of a highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amylovora in asymptomatic plant material.

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 microl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.