Single Primer Pair Research Articles

Simultaneous TD/RT-PCR detection of cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus with a single pair of primers

Cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) are the two most prevalent and economically important orchid viruses which have attained a world-wide distribution. We have designed one pair of common primers, which were able to detect, simultaneously, both viruses by a single polymerase chain reaction (PCR). Sequence alignment and primer analyses were employed in primer design. The effect of touch-down PCR, primer annealing temperature, number of thermal cycles, MgCl 2 concentration and the degree of primer degeneracy, were investigated and applied to the optimisation of PCR conditions in the detection of both viruses in order to achieve equal amplification efficiencies. This is the first successful attempt at using a single pair of PCR primers to detect two viruses belonging to different taxonomic groups simultaneously.

Development of a multiplex-PCR for direct detection of the genes for enterotoxin B and C, and toxic shock syndrome toxin-1 in Staphylococcus aureus isolates.

As well as conventional methods such as immunodiffusion, ELISA, or agglutination for the detection of toxin production in Staphylococcus aureus, amplification techniques like PCR allow a very sensitive and specific identification of the genes responsible for enterotoxin B and C, and TSST-1 production. These toxins might be a cause of the toxic shock syndrome (TSS). For that reason an easy and quick test system for determining the toxin production pattern of S. aureus isolates is desirable so that strains suspected to be toxin producers may be identified much faster and easier. In the present investigation, a new multiplex-PCR method was used that allowed single bacterial colonies grown on agar plates to be used directly in the PCR assay without preceding preparation. This procedure generated information concerning the presence of seb, sec-1 and tst genes within 4 h in a single test. To analyse the sensitivity and the specificity of this procedure, 100 methicillin-resistant S. aureus (MRSA), 50 coagulase-negative staphylococci and 50 other eubacterial isolates were tested initially with sets of single primer pairs followed by a combined multiplex-PCR. Results of this amplification technique were compared to a conventional and widely used method for toxin detection, reversed passive latex agglutination (RPLA). With the RPLA assay results as the basis, sensitivity and specificity of the seb and tst primer sets were 100%, whereas sensitivity and specificity of the sec-1 primer set were 100% and 82%, respectively. With the sec-1 primer set, two isolates were identified as carrying the corresponding toxin gene although the RPLA test did not show any detectable toxin. The multiplex-PCR rapidly generated reliable information concerning the toxin-producing capacity of staphylococcal strains and could be easily integrated into a multiplex procedure described previously. The latter enabled the identification of specific PCR products for eubacteria and staphylococci as well as the detection of the coa and mecA genes.

Separation of Slovenian isolates of PVY NTN from the North American isolates of PVY N by a 3-primer PCR

The potato tuber ringspot necrosis isolate of potato virus Y (PVY NTN) is a recently recognized and highly aggressive isolate of the PVY N group of strains. In order to screen specifically sources of resistance to PVY NTN a method to separate PVY NTN from PVY N is needed. To achieve this, 61 isolates from 13 imported and locally developed potato cultivars in Slovenia were studied. On the basis of the reactions in indicator plants Nicotiana tabacum cv. Samsun and Solanum brachycarpum and with a PVY N specific monoclonal antibody (4E7), all Slovenian isolates (Sl-NTN) were identified as PVY N. Using two primer pairs from the P1 gene of a Hungarian isolate of PVY NTN by a conventional single primer pair, reverse transcription polymerase chain reaction (RT-PCR) both PVY NTN and PVY N were amplified similarly. However, specific amplification of PVY NTN was achieved by a nested-PCR at an annealing temperature of 63°C. A simplified form of the nested-PCR, termed 3-primer PCR was developed, which is applicable for large-scale testing of samples. Using the 3-primer PCR at annealing temperature of 63°C, known mixtures of PVY NTN and PVY N were correctly separated. PVY NTN was detected in dormant tubers and leaves from all Sl-NTN isolates. The 3-primer PCR was specific to PVY NTN and did not react with nine isolates of PVY N, 13 isolates of PVY o, one isolate of PVY C, six commonly occurring potato viruses and a viroid.

Heterogeneity in Bacillus cereus PCR products detected by ESI-FTICR mass spectrometry.

PCR amplification of a segment of the 16/23S rDNA interspace region (ISR) from Bacillus cereus 6464 produced a mixture of products. An 89-bp product was predicted on the basis of the reported sequence. The ESI-FTICR analysis revealed three double-stranded products, differing in size by a single nucleotide corresponding to two homoduplexes of 89 and 88 base pairs and a heteroduplex of 89 and 88 nucleotide strands. These were produced from a single preparation of genomic DNA and a single primer pair. ESI-FTICR analysis of the single strands identified a deletion of a T in the coding strand and a corresponding loss of an A in the noncoding strand of this product. The ESI-FTICR analysis indicated the presence of an unreported sequence variation between rRNA operons in this organism. This report illustrates that PCR products amplified from templates differing by a single nucleotide can be resolved and identified using ESI-FTICR at the 89-bp level. Furthermore, the ESI-FTICR mass measurements provided the identity of the deletion, which is indicative of interoperon variability.

Rapid detection of Candida albicans in clinical samples by DNA amplification of common regions from C. albicans-secreted aspartic proteinase genes.

Laboratory diagnosis based on genomic amplification methods such as PCR may provide an alternative and more sensitive method than conventional culture for the early detection of deep-seated candidiasis, an increasing cause of morbidity and mortality among immunocompromised patients. A novel method of DNA extraction from clinical samples based on treatment with proteinase K and isolation of DNA on a silica membrane was developed. The targets used for DNA amplification were the Candida albicans-secreted aspartic proteinase (SAP) genes, a multiple-gene family of at least seven members in C. albicans. A single pair of primers was designed in order to detect six of these SAP genes and, subsequently, to increase the sensitivity of the test. Detection of the PCR product by enzyme-linked immunosorbent assay was found to be as sensitive as Southern blotting with an SAP-labeled probe. The sensitivity of the assay was 1 cell/ml from serially diluted Candida cultures and 1 to 4 cells/ml from seeded blood specimens. The sensitivity and specificity of the present assay were tested in a retrospective study performed blindly with 156 clinical samples and were 100 and 98%, respectively, compared with the results of culture. For the subset of blood culture samples (n = 124), the sensitivity and the specificity were 100%. The two false-positive PCR samples came from patients treated with azole antifungal agents, indicating that PCR was probably able to detect damaged organisms that could not be recovered by culture.

An Evaluation of the AFLP Fingerprinting Technique for the Analysis of Paternity in Natural Populations of Persoonia mollis (Proteaceae)

The accurate assignment of paternity in natural plant populations is required to address important issues in evolutionary biology, such as the factors that affect reproductive success. Newly developed molecular fingerprinting techniques offer the potential to address these aims. Here, we evaluate the utility of a new PCR-based multi-locus fingerprinting technique called Amplified Fragment Length Polymorphism (AFLP) for paternity studies in Persoonia mollis (Proteaceae). AFLPs were initially scored for five individuals from three taxonomic levels for 64 primer pairs: between species (P. mollis and P. levis), between subspecies (P. mollis subsp. nectens and subsp. livens), between individuals within a single population of P. mollis, as well as for a naturally pollinated seed from a single P. mollis subsp. nectens plant. Overall, 1164 fragments (24.6% of all fragments) were polymorphic between species, 743 (16.5%) between subspecies, 371 (8.6%) between individuals within a single population, and 265 (6.2%) between a plant and its seed. Within a single P. mollis population of 14 plants, 42 polymorphic fragments were scored from profiles generated by a single AFLP primer pair. The mean frequency of the recessive allele (q) over these 42 loci was 0.773. Based on these observations, it will be feasible to generate well over 100 polymorphic AFLP loci with as few as three AFLP primer pairs. This level of polymorphism is sufficient to assign paternity unambiguously to more than 99% of all seed in experiments involving small, known paternity pools. More generally, the AFLP procedure is well suited to molecular ecological studies, because it produces more polymorphism than allozymes or RAPDs but, unlike conventionally developed microsatellite loci, it requires no prior sequence knowledge and minimal development time.

Cell type-specific inhibition of keratinocyte collagenase-1 expression by basic fibroblast growth factor and keratinocyte growth factor. A common receptor pathway.

Collagenase-1 is invariantly expressed by migrating basal keratinocytes in all forms of human skin wounds, and its expression is induced by contact with native type I collagen. However, net differences in enzyme production between acute and chronic wounds may be modulated by soluble factors present within the tissue environment. Basic fibroblast growth factor (bFGF, FGF-2) and keratinocyte growth factor (KGF, FGF-9), which are produced during wound healing, inhibited collagenase-1 expression by keratinocytes in a dose-dependent manner. However, KGF was >100-fold more effective than bFGF at inhibiting collagenase-1 expression, suggesting that this differential signaling is transduced via an FGF receptor that binds these ligands with different affinities. Reverse transcriptase-polymerase chain reaction analysis of human keratinocyte mRNA for fibroblast growth factor receptors (FGFRs) revealed expression of only FGFR-2 IIIb, the KGF-specific receptor, which also binds bFGF with low affinity, and FGFR-3 IIIb, which does not bind bFGF or KGF. FGFRs that bind bFGF with high affinity were not detected. Our results suggest that bFGF and KGF inhibit collagenase-1 expression through the KGF cell-surface receptor (FGFR-2 IIIb). Because bFGF induces collagenase-1 in most cell types, cell-specific expression of FGFR family members may dictate the regulation of matrix metalloproteinases in a tissue-specific manner.

Gene for A-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses

Orthopoxvirus species were identified and differentiated by polymerase chain reaction amplification of genome DNA using a single primer-pair based on sequences coding for the major protein component of the cowpox virus acidophilic-type inclusion body (ATI). DNA available for 6 of 8 Old World (cowpox, variola, monkeypox, camelpox, ectromelia and vaccinia viruses) and 3 New World (skunkpox, volepox, and raccoonpox) resulted in amplicons that ranged in size from 510 to 1673 base pairs depending on the species, except for raccoonpox virus DNA which did not amplify. XbaI digest gel electrophoresis profiles of the amplicons improved resolution of the differences.

Geographic differences in the sensitivity of a polymerase chain reaction for the detection of Plasmodium falciparum infection.

The amplification of target DNA by highly specific probes using the polymerase chain reaction (PCR) provides a highly sensitive and specific method for the detection of malaria infection. The use the of PCR in settings with varying endemicity within one survey area has not been investigated intensively. Therefore, a cross-sectional study was conducted in the districts of Kabarole and Bundibugyo in western Uganda using material from three villages with different epidemiologic situations regarding malaria and DNA primers for a PCR that had shown satisfactory sensitivity and specificity in previous trials. The sensitivity of the PCR varied significantly (P < 0.001) in the three survey villages (between 63.2% and 83.9% for the primer pair K1-14-1 and between 37.9% and 69.9% for the primer pair MSP-1) and was highly linked to geographic differences and social exchanges of the inhabitants with other areas of the district. According to the results of this investigation, it is advisable not to use a single primer pair in epidemiologic field studies for the detection of falciparum malaria. The use of combined primer pairs and the frequent confirmation of the results by microscopy are recommended.

Detection of BCR-abl in acute leukemia by molecular and cytogenetic methods

Background: The Philadelphia chromosome (Ph), t(9;22)(q34;q11), is detected by karyotyping in a minority of patients with acute leukemia. Ph results in fusion of the c-abl oncogene on chromosome 9 with the breakpoint cluster region BCR gene on chromosome 22. The purpose of this study was to compare reverse trascriptase-polymerase chain reaction (RT-PCR) for BCR/abl fusion to cytogenetic methods for Ph detection in patients with acute leukemia. Methods and Results: Peripheral blood and bone marrow samples from cases of adult acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) were examined for Ph by RT-PCR, karyotyping, and fluorescence in situ hybridization (FISH). Using total cellular RNA and a single primer pair, cDNA was transcribed, amplified, electorphoresed, and probed for BCR/abl fusion. Patient cells and SUPB15 and K562 cell lines were used as breakpoint controls. Karyotyping was done by standard Giemsa banding. FISH was performed on bone marrow smears using digxigenin-labeled DNA probes for major and minor bcr breakpoints (corresponding to involvement of major bcr exons 2/3 and minor bcr exon 1, respectively) and biotin-labeled DNA probes for abl. Rhodamine-conjugated antidigoxigenin and fluorescein-conjugated avidin yielded red and green fluorescent signals, respectively. A total of 32 samples from patients with AML were studied, 20 from patients with de novo or relapsed AML and 12 from patients in remission. Five of 32 cases of AML (16%) were RT-PCR+/Ph+ all with major bcr breakpoints between exons 3 and 4. One of the 32 cases (3%) was RT-PCR+/Ph+; this case was the only positive remission sample. Of the four T-PCR+/Ph- cases, one showed t(2;17), one showed t(9;11), and two had a normal karyotype. FISH was done in three RT-PCR+ cases, yielding positive results with the major probe in two. A total of 22 samples from patients with ALL were studies, 15 from patients with de novo or relapsed ALL and seven from patient remission. Seven of 22 cases of ALL (32%) were RT-PCR+, four with major-bcr breakpoints between exons 3 and 4, and three with breakpoints in the minor-bcr. Two of the 22 (9%) cases were RT- PCR+/Ph+. Of the five RT-PCR+/Ph- cases, two showed a 22q- but lacked the typical Ph break on chromosome 9, 1 showed a 12p-, and two had a normal karyotype. FISH was performed in four RT-PCR+ cases, yielding positive results with the major probe in two cases and with the minor probe in two cases. Conclusions: RT-PCR is more sensitive than karyotyping, detecting masked Ph or translocations not found by cytogenetic analysis. FISH is a helpful adjunctive test when used to confirm BCR/abl fusion in RT-PCR+/Ph- cases. (Mol Diagn 1996 Dec;1(4):305-313)

Analysis of loss of heterozygosity in microdissected tumor cells from cervical carcinoma using fluorescent dUTP labeling of PCR products.

The use of fluorescently labeled nucleotides (F-dUTP) in the PCR for genetic typing of microsatellite polymorphisms was investigated. Microdissected tumor cells from cervical squamous cancer biopsies were compared with cells from surrounding normal tissue for loss of heterozygosity (LOH) at the mismatch repair gene hMLH1. Removal of unincorporated F-dUTP before analysis is necessary to reduce background fluorescence; ethanol-precipitation was found to be as efficient as the use of a spin column for this purpose. The gel resolution was sufficient to distinguish alleles differing by about four nucleotides. Alleles differing by only one dinucleotide repeat were possible to identify, but the ratio of alleles was difficult to assert with any reliability due to the wide peaks obtained. Single primer-pair and nested amplification systems were shown in reconstitution experiments to reliably quantitate the ratio of alleles. Of 36 cervical cancer biopsies, amplification and typing of the hMLH1 microsatellite marker was successful in 20 cases. Among 9 informative (heterozygous) biopsies, 2 (22%) were found to show LOH.

Regenerating (reg) and insulin genes are expressed in prepancreatic mouse embryos

The pancreatic regenerating (reg) gene is proposed to be involved in pancreatic beta-cell growth. Up- or down-regulation of reg gene expression has been shown to parallel variations in beta-cell mass and function in the adult pancreas. In several species at least two nonallelic reg genes have been identified. In this study we investigated the expression of each individual reg gene (reg-I and reg-II) during embryogenesis in the mouse. Single mouse embryos were harvested at 8.5, 9, 10, and 12 days of development, homogenized and subjected individually to reverse transcription (RT)-PCR, with a single primer pair to amplify both reg-I and -II mRNAs. Southern blot analysis of the RT-PCR products revealed the presence of reg mRNA at day 9 of embryogenesis, just before the beginning of pancreatic organogenesis. Slot-blot analysis with internal oligonucleotide probes that specifically recognize reg-I or -II sequences demonstrated that only reg-I mRNA was present in day 9 and day 10 prepancreatic embryos. Reg-II mRNA was not detected until day 12, a stage corresponding to late organogenesis. RT-PCR for insulin mRNA from the same samples used for the amplification of reg mRNA showed that the earliest insulin expression occurred at day 8.5, and coincided with the onset of reg-I expression. Hybridization with gene-specific oligonucleotide probes revealed that only insulin-II mRNA was detectable at this time. Insulin-I mRNA was not detectable until day 12 and coincided with early reg-II expression. These results suggest that the two nonallelic reg genes and the two insulin genes are expressed differentially during early embryogenesis. Differential expression of reg-I and -II suggests that they may be induced by different and independent stimuli and have distinct functions.

A PCR assay for detection of a 2RL.2BS wheat-rye chromosome translocation.

A 2RL.2BS wheat-rye translocation, present in the wheat germplasm line Hamlet, carries a gene for resistance to Hessian fly biotype L, one of the most virulent biotypes presently encountered in wheat production environments. Unlike several other wheat-rye chromosome translocations common in wheat breeding programs, 2RL lacks genes encoding storage proteins or other easily selected markers. Oligonucleotide primers synthesized from published sequences derived from the R173 family of moderately repetitive rye DNA were used in the DNA polymerase chain reaction (PCR) to identify specific markers for 2RL. The same primers, when used with DNA extracted from additional wheat-rye translocation lines of importance to the wheat breeding community, gave distinctive PCR products for each genotype. The single primer pair, PAWS5 and PAWS6, may, therefore, have wide applicability for the identification of wheat-rye chromosomal translocations presently encountered in wheat breeding populations.

Direct in situ reverse transcriptase polymerase chain reaction for detection of measles virus.

New methods are described for combined intracellular reverse transcription (RT) and polymerase chain reaction (PCR) using single primer pairs, with direct incorporation of digoxigenin-11-dUTP into amplificants (direct in situ RT/PCR). Routinely used fixatives and minimal pre-treatments were employed. Target sequences of measles virus nucleocapsid (N) and phosphoprotein genes were detected within measles virus infected Vero cells, both in suspension and in formalin-fixed sections, that had been treated by in situ reverse transcription and 30 cycles of direct in situ PCR. Uninfected cells, omission of Taq polymerase, and irrelevant primers were used as controls. Distribution of measles virus within infected cells was determined by in situ hybridisation and immunocytochemistry for measles virus N gene and protein, respectively. Confirmation of amplification within sections was by gel electrophoresis, Southern blotting and sequencing of extracted amplicons. In the majority of cases, measles-infected cells exhibited intense cytoplasmic signal after direct in situ PCR; this was not seen in uninfected cells or infected cells reacted either with irrelevant primers or without Taq polymerase. Unfixed cells in suspension required nested reaction. Measles-specific in situ hybridisation and immunocytochemistry gave an identical signal distribution in sections. Nuclear artifact occurred in some sections and was unpredictable, although it was greatest either in areas of cellular damage, following DNase predigestion, or with vigorous protease pre-treatment. In situ RT-PCR is feasible for measles virus in acutely infected cells both in sections and in suspension. Further work is required to improve the procedure and to eliminate artefactual nuclear signal.

Reverse Transcriptase-Polymerase Chain Reaction forbcr/ablFusion in Chronic Myelogenous Leukemia

Reverse transcriptase-polymerase chain reaction (RT-PCR) has shown promise as a means of detecting low levels of cells bearing the Philadelphia chromosome (Ph1) and for detecting cytogenetically inapparent ("masked") Ph1 in patients with chronic myelogenous leukemia (CML). For detection by karyotyping, dividing cells must be used, precluding use of peripheral blood samples in cases with low peripheral blood blast counts. Reverse transcriptase-polymerase chain reaction was performed in 83 bone marrow and 30 peripheral blood samples from patients with CML to compare results with karyotyping and to evaluate utility of this test on peripheral blood samples. Using isolated total cellular RNA and a single primer pair, cDNA was transcribed, amplified, electrophoresed, and probed for bcr/abl fusion involving M-bcr exons 2 and 3 of the bcr gene. Fifty-three samples were from untreated or conventionally treated patients (pre-BMT), and 60 were from patients who had undergone bone marrow transplantation (post-BMT). Fifty of 53 pre-BMT samples were positive by RT-PCR. Two samples, negative by RT-PCR, had complex translocations, t(9;16;22) and t(4;14;22). One case was indeterminate by RT-PCR, but positive on retesting. Forty-five of 53 had Ph1 by karyotyping; 8 were negative, including 5 peripheral blood samples, 2 bone marrow samples with "masked" Ph1, and 1 bone marrow sample with poor growth. Thirty-five of 60 post-BMT samples were positive by RT-PCR. Fourteen of 60 post-BMT samples had Ph1 by karyotyping. Of the RT-PCR+/Ph1- cases, most showed a weak but definite band by RT-PCR, suggesting a low level of the bcr/abl fusion gene. Nineteen patients had concurrent peripheral blood and bone marrow samples analyzed by RT-PCR and karyotyping. Of 16 patients with satisfactory RNA extraction, 15 had concordant results by RT-PCR. Five patients had adequate metaphase cells for karyotypic analysis. All had Ph1 in bone marrow, but were negative in peripheral blood. Our results indicate that RT-PCR for detection of bcr/abl fusion is more sensitive than karyotyping in pre- and post-BMT samples. Furthermore, RT-PCR can be successfully performed on peripheral blood, yielding excellent correlation with bone marrow samples.

Microsatellite analysis of relationships within cultivated potato (Solanum tuberosum)

The potential of microsatellite markers for use in genetical studies in potato (Solanum tuberosum) was evaluated. Database searches revealed that microsatellite sequences were present in the non-coding regions of 24 potato genes. Twenty-two sets of primers were designed and products successfully amplified using 19 primer pairs. These were tested against a panel of 18 tetraploid potato cultivars. Four pairs of primers designed to amplify microsatellites from tomato were also used. Seven (including 2 of the tomato sequences) failed to reveal any variation in the accessions tested. Sixteen primer pairs did reveal polymorphism, detecting between 2 and 19 alleles at each locus. Of these, 3 gave rise to complex band patterns, suggesting that multiple polymorphic loci were being amplified using a single primer pair. Heterozygosity values ranged from 0.408 to 0.921. Phenetic analysis of the derived information allowed a dendrogram to be constructed depicting the relationships between the 18 potato cultivars. The potential of microsatellite markers for genetic analysis and satutory applications in potato is discussed in the context of these results. Furthermore, the potential of 'crossspecies amplification' is highlighted as an additional source of microsatellite markers for genetic research in potato.

Optimal primer selection for clonality assessment by polymerase chain reaction analysis III. Intermediate and high-grade B-cell neoplasms

Previous studies have reported that low-grade B-cell lymphoproliferative disorders have variable B-cell monoclonality detection rates by polymerase chain reaction (PCR) analysis. For instance, monoclonal B-cell populations from chronic lymphocytic leukemia/small lymphocytic leukemia and mantle cell lymphoma are most often readily amplified with a single primer pair, whereas follicular lymphomas and plasma cell neoplasms require alternative strategies to approach these higher diagnostic sensitivity standards. Because most published reports have not focused on the variation in PCR B-cell monoclonality detection among subtypes of intermediate and high-grade B-cell neoplasms, additional information is necessary to determine primer selection strategies and identify problematic tumor subtypes within this group. The current investigation, the third part in a series, was aimed at documenting the efficiency of B-cell monoclonality detection by PCR in 71 aggressive B-cell neoplasms of various types using a comprehensive approach. A predetermined panel of primer sets was used in an algorithmic fashion. Specifically, all samples were analyzed with the standard VH-FRIII/JH assay previously shown to have the highest efficiency of monoclonality detection within low-grade B-cell lymphoproliferative disorders. Negative samples were further evaluated with primer sets in the following order until a positive result was observed, or all primer sets were used: (1) bcl-2/JH, (2) VH-FRI family specific/JH, and (3) VH-FRI consensus/JR. Forty-three (61%) of the 71 B-cell neoplasms evaluated with VH-FRIII/JH showed monoclonal B-cell populations. Sequential use of the three reserve primer sets in samples negative with this initial primer pair resulted in an overall improvement in PCR detection from 61% to 82% (58 of 71 specimens) ( P < .001). The VH-FRI family specific assay identified B-cell monoclonality in 11 (73%) of these 15 specimens and was the most productive reserve primer set. Individual categories exhibited the following initial (I) and final (F) PCR detection rates: acute lymphoblastic leukemia/ lymphoblastic lymphoma, 11 specimens (I = 91% to F = 91%); small noncleaved cell lymphoma, 14 specimens (I = 79% to F = 86% [ P > .25]); diffuse large cell lymphoma, 33 specimens (I = 52% to F = 85% [ P < .005]) and large cell, immunoblastic lymphoma, 13 specimens (I = 38% to F = 62% [ P < .01]). The authors have shown that comprehensive PCR analysis is capable of detecting B-cell monoclonality in a significant proportion of samples from each subtype of intermediate and high-grade B-cell neoplasm. The VH-FRIII/JH assay was an adequate initial primer set, but required augmentation with the reserve PCR assays to attenuate the false negative rate and improve diagnostic sensitivity. The B-cell clonality PCR assay is optimally used as a screening tool and when used in this fashion, the more laborious and time-consuming restriction fragment-Southern blot hybridization (RF-SBH) method for IgH gene rearrangement detection may be limited to a relatively small proportion of PCR-negative aggressive B-cell neoplasms.

Study of the intergenic exeF-exeG region and its application as a simple preliminary test for Aeromonas spp.

The exeF-exeG intergenic regions from different hybridization groups (HG) of Aeromonas were studied by PCR amplification using a single pair of primers. Six main classes of PCR products were identified according to size: 360 bp, 320 bp, 280 bp, 230–240 bp, 220 bp and 160 bp. Direct sequencing of the PCR products indicated that the shorter intergenic regions had probably originated from deletion of DNA segments between direct repeats. Correlation of certain PCR products with Aeromonas caviae (HG4), A. caviae (HG5), A. veronii (HG8) and A. salmonicida (HG3) was revealed. The PCR reaction was also shown to be generally specific for Aeromonas spp. Thus, the usefulness of this rapid, single colony-based PCR test for both identification and preliminary differentiation of Aeromonas spp. is demonstrated.

Study of the intergenic exeF-exeG region and its application as a simple preliminary test for Aeromonas spp.

The exeF-exeG intergenic regions from different hybridization groups (HG) of Aeromonas were studied by PCR amplification using a single pair of primers. Six main classes of PCR products were identified according to size: 360 bp, 320 bp, 280 bp, 230-240 bp, 220 bp and 160 bp. Direct sequencing of the PCR products indicated that the shorter intergenic regions had probably originated from deletion of DNA segments between direct repeats. Correlation of certain PCR products with Aeromonas caviae (HG4), A. caviae (HG5), A. veronii (HG8) and A. salmonicida (HG3) was revealed. The PCR reaction was also shown to be generally specific for Aeromonas spp. Thus, the usefulness of this rapid, single colony-based PCR test for both identification and preliminary differentiation of Aeromonas spp. is demonstrated.

Creation of deletion, insertion and substitution mutations using a single pair of primers and PCR.

BioTechniquesVol. 20, No. 2 BenchmarksOpen AccessCreation of Deletion, Insertion and Substitution Mutations Using a Single Pair of Primers and PCRMartin John Glenton Hughes & David William AndrewsMartin John Glenton HughesMcMaster University, Hamilton, ON, CanadaSearch for more papers by this author & David William Andrews*Address correspondence to David W. Andrews, McMaster University, Department of Biochemistry, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada.McMaster University, Hamilton, ON, CanadaSearch for more papers by this authorPublished Online:2 Aug 2018https://doi.org/10.2144/96202bm06AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByA Syringe‐Based DNAzyme Sensor for Bacterial Detection7 April 2021 | Analysis & Sensing, Vol. 1, No. 2In Vitro Mutagenesis Using Double-Stranded DNA Templates: Selection of Mutants with DpnI1 March 2018 | Cold Spring Harbor Protocols, Vol. 2018, No. 3A Mutant dec-1 Transgene Induces Dominant Female Sterility in Drosophila melanogaster1 November 2007 | Genetics, Vol. 177, No. 3Genomic structure and functional characterization of the promoter region of human IκB kinase-related kinase IKKi/IKKɛ geneGene, Vol. 353, No. 1The N-terminal prodomain of sV23 is essential for the assembly of a functional vitelline membrane network in DrosophilaDevelopmental Biology, Vol. 270, No. 1A Direct Binding Site for Grb2 Contributes to Transformation and Leukemogenesis by the Tel-Abl (ETV6-Abl) Tyrosine KinaseMolecular and Cellular Biology, Vol. 24, No. 11Active site residues and mechanism of UDP-glucose dehydrogenase14 December 2003 | European Journal of Biochemistry, Vol. 271, No. 1Mutational Analysis of the TonB1 Energy Coupler of Pseudomonas aeruginosaJournal of Bacteriology, Vol. 184, No. 6The Functions of the Multiproduct and Rapidly Evolving dec-1 Eggshell Gene Are Conserved Between Evolutionarily Distant Species of Drosophila1 November 2001 | Genetics, Vol. 159, No. 3gmhX, a novel gene required for the incorporation of l-glycero-d -manno-heptose into lipooligosaccharide in Neisseria meningitidis The GenBank accession number for the sequence of the ice-2 operon in Neisseria meningitidis NMB is AF036242.Microbiology, Vol. 147, No. 8Nucleotide-dependent Binding of the GTPase Domain of the Signal Recognition Particle Receptor β-Subunit to the α-SubunitJournal of Biological Chemistry, Vol. 275, No. 35Spatial and Temporal Expression of a Polysphondylium Spore-Specific GeneDevelopmental Biology, Vol. 224, No. 1Evidence that the 5′-End Cap Structure Is Essential for Encapsidation of Hepatitis B Virus Pregenomic RNAJournal of Virology, Vol. 74, No. 12Modulation of casein proteolysis by lactococcal peptidase gene inactivationInternational Dairy Journal, Vol. 10, No. 9A Site-specific, Membrane-dependent Cleavage Event Defines the Membrane Binding Domain of FtsYJournal of Biological Chemistry, Vol. 274, No. 47Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil DifferentiationBlood, Vol. 93, No. 12Multiple lysophosphatidic acid acyltransferases in Neisseria meningitidisMolecular Microbiology, Vol. 32, No. 5Improved Protocol for High-Throughput Cysteine Scanning MutagenesisStefan Howorka & Hagan Bayley15 August 2018 | BioTechniques, Vol. 25, No. 5Endothelin Converting-Enzyme-1 mRNA Expression in Human Cardiovascular Disease3 July 2009 | Clinical and Experimental Hypertension, Vol. 20, No. 4Modification of a PCRBased Site-Directed Mutagenesis MethodConstance L. Fisher & Guo Kui Pei7 August 2018 | BioTechniques, Vol. 23, No. 4Transcriptional Induction of Collagenase-1 in Differentiated Monocyte-like (U937) Cells is Regulated by AP-1 and an Upstream C/EBP-β SiteJournal of Biological Chemistry, Vol. 272, No. 18Primer-Directed Mutagenesis of an Intact Plasmid by Using Pwo DNA Polymerase in Long Distance Inverse PCRRachmat Hidajat & Patricia McNicol7 August 2018 | BioTechniques, Vol. 22, No. 1Site-Directed Mutagenesis for Large Insertions by Oligonucleotide Primers in Optimized Molar RatiosDong Wang & Hei Sook Sul7 August 2018 | BioTechniques, Vol. 22, No. 1 Vol. 20, No. 2 Follow us on social media for the latest updates Metrics Downloaded 381 times History Published online 2 August 2018 Published in print February 1996 Information© 2018 Author(s)PDF download