The article describes a new technology for real-time polymerase chain reaction (PCR) detection of nucleic acids. Similar to Taqman, this new method, named Snake, utilizes the 5′-nuclease activity of Thermus aquaticus (Taq) DNA polymerase that cleaves dual-labeled Förster resonance energy transfer (FRET) probes and generates a fluorescent signal during PCR. However, the mechanism of the probe cleavage in Snake is different. In this assay, PCR amplicons fold into stem–loop secondary structures. Hybridization of FRET probes to one of these structures leads to the formation of optimal substrates for the 5′-nuclease activity of Taq. The stem–loop structures in the Snake amplicons are introduced by the unique design of one of the PCR primers, which carries a special 5′-flap sequence. It was found that at a certain length of these 5′-flap sequences the folded Snake amplicons have very little, if any, effect on PCR yield but benefit many aspects of the detection process, particularly the signal productivity. Unlike Taqman, the Snake system favors the use of short FRET probes with improved fluorescence background. The head-to-head comparison study of Snake and Taqman revealed that these two technologies have more differences than similarities with respect to their responses to changes in PCR protocol, e.g. the variations in primer concentration, annealing time, PCR asymmetry. The optimal PCR protocol for Snake has been identified. The technology’s real-time performance was compared to a number of conventional assays including Taqman, 3′-MGB-Taqman, Molecular Beacon and Scorpion primers. The test trial showed that Snake supersedes the conventional assays in the signal productivity and detection of sequence variations as small as single nucleotide polymorphisms. Due to the assay’s cost-effectiveness and simplicity of design, the technology is anticipated to quickly replace all known conventional methods currently used for real-time nucleic acid detection.
Scorpion primers can be used to detect PCR products in homogeneous solution. Their structure promotes a unimolecular probing mechanism. We compare their performance with that of the same probe sequence forced to act in a bimolecular manner. The data suggest that Scorpions indeed probe by a unimolecular mechanism which is faster and more efficient than the bimolecular mechanism. This mechanism is not dependent on enzymatic cleavage of the probe. A direct comparison between Scorpions, TaqMan and Molecular Beacons on a Roche LightCycler indicates that Scorpions perform better, particularly under fast cycling conditions. Development of a cystic fibrosis mutation detection assay shows that Scorpion primers are selective enough to detect single base mutations and give good sensitivity in all cases. Simultaneous detection of both normal and mutant alleles in a single reaction is possible by combining two Scorpions in a multiplex reaction. Such favourable properties of Scorpion primers should make the technology ideal in numerous applications.
In order to establish an accurate, ready-to-use assay for simultaneous detection of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV), we developed one duplex TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay, which can be used in human and vector surveillance. First, we selected the primers and FAM-labeled TaqMan-probe specific for WEEV from the consensus sequence of NSP3 and the primers and HEX-labeled TaqMan-probe specific for EEEV from the consensus sequence of E3, respectively. Then we constructed and optimized the duplex real-time RT-PCR assay by adjusting the concentrations of primers and probes. Using a series of dilutions of transcripts containing target genes as template, we showed that the sensitivity of the assay reached 1 copy/reaction for EEEV and WEEV, and the performance was linear within the range of at least 106 transcript copies. Moreover, we evaluated the specificity of the duplex system using other encephalitis virus RNA as template, and found no cross-reactivity. Compared with virus isolation, the gold standard, the duplex real time RT-PCR assay we developed was 10-fold more sensitive for both WEEV and EEEV detection.
Enterovirus (EV) infections are commonly associated with encephalitis and meningitis. Detection of enteroviral RNA in clinical specimens has been demonstrated to improve the management of patients, by ruling out other causes of disease.
To develop a sensitive and reliable assay for routine laboratory diagnosis, we developed a real-time one step reverse transcription polymerase chain reaction (RT-PCR) assay with minor groove binder probes and primers modified with complementary locked primer technology (TMC-PCR). We checked the sensitivity of the developed assay by comparing it to a previously published TaqMan probe real-time one-step RT-PCR (TTN-PCR) procedure using enteroviral isolates, Enterovirus Proficiency panels from Quality Control on Molecular Diagnostics (QCMD-2007), and clinical specimens from patients with suspected EV infections.
One hundred clinical specimens from 158 suspected viral meningitis cases were determined to be positive by the TMC-PCR assay (63.29%), whereas only 60 were found to be positive by the TTN-PCR assay (37.97%). The positive and negative agreements between the TMC-PCR and TTN-PCR assays were 100% and 59.2%, respectively.
This data suggest that the TMC-PCR assay may be suitable for routine diagnostic screening from patient suspected EV infection.
Aseptic meningitis; Real-time one step RT-PCR; CLP; MGB probe
A TaqMan-based real-time polymerase chain reaction (PCR) assay was devised for the detection of porcine parvovirus (PPV). Two primers and a TaqMan probe for the non-structural protein NS1 gene were designed. The detection limit was 1 × 102 DNA copies/μL, and the assay was linear in the range of 1 × 102 to 1 × 109 copies/μL. There was no cross-reaction with porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The assay was specific and reproducible. In 41 clinical samples, PPV was detected in 32 samples with the real-time PCR assay and in only 11 samples with a conventional PCR assay. The real-time assay using the TaqMan-system can therefore be practically used for studying the epidemiology and management of PPV.
The real-time polymerase chain reaction is currently the method of choice for quantifying nucleic acids in different DNA based quantification applications. It is widely used also for detecting and quantifying genetically modified components in food and feed, predominantly employing TaqMan® and SYBR® Green real-time PCR chemistries. In our study four alternative chemistries: Lux™, Plexor™, Cycling Probe Technology and LNA® were extensively evaluated and compared using TaqMan® chemistry as a reference system.
Amplicons were designed on the maize invertase gene and the 5'-junction of inserted transgene and plant genomic DNA in MON 810 event. Real-time assays were subsequently compared for their efficiency in PCR amplification, limits of detection and quantification, repeatability and accuracy to test the performance of the assays. Additionally, the specificity of established assays was checked on various transgenic and non-transgenic plant species. The overall applicability of the designed assays was evaluated, adding practicability and costs issues to the performance characteristics.
Although none of the chemistries significantly outperformed the others, there are certain characteristics that suggest that LNA® technology is an alternative to TaqMan® when designing assays for quantitative analysis. Because LNA® probes are much shorter they might be especially appropriate when high specificity is required and where the design of a common TaqMan® probe is difficult or even impossible due to sequence characteristics. Plexor™ on the other hand might be a method of choice for qualitative analysis when sensitivity, low cost and simplicity of use prevail.
Molecular beacon probes can be employed in a NASBA amplicon detection system to generate a specific fluorescent signal concomitantly with amplification. A molecular beacon, designed to hybridize within the target sequence, was introduced into NASBA reactions that amplify the genomic RNA of potato leafroll virus (PLRV). During amplification, the probe anneals to the antisense RNA amplicon generated by NASBA, producing a specific fluorescent signal that can be monitored in real-time. The assay is rapid, sensitive and specific. As RNA amplification and detection can be carried out in unopened vessels, it minimizes the risk of carry-over contaminations. Robustness has been verified on real-world samples. This homogeneous assay, called AmpliDet RNA, is a significant improvement over current detection methods for NASBA amplicons and is suitable for one-tube applications ranging from high-throughput diagnostics to in vivo studies of biological activities.
We have developed a hepatitis B virus (HBV) DNA detection and quantification system based on amplification with nucleic acid sequence-based amplification (NASBA) technology and real-time detection with molecular beacon technology. NASBA is normally applied to amplify single-stranded target RNA, producing RNA amplicons. In this work we show that with modifications like primer design, sample extraction method, and template denaturation, the NASBA technique can be made suitable for DNA target amplification resulting in RNA amplicons. A major advantage of our assay is the one-tube, isothermal nature of the method, which allows high-throughput applications for nucleic acid detection. The homogeneous real-time detection allows a closed-tube format of the assay, avoiding any postamplification handling of amplified material and therefore minimizing the risk of contamination of subsequent reactions. The assay has a detection range of 103 to 109 HBV DNA copies/ml of plasma or serum (6 logs), with good reproducibility and precision. Compared with other HBV DNA assays, our assay provides good sensitivity, a wide dynamic range, and high-throughput applicability, making it a viable alternative to those based on other amplification or detection methods.
Identification of Anopheles gambiae s.s. and Anopheles arabiensis from field-collected Anopheles gambiae s.l. is often necessary in basic and applied research, and in operational control programmes. The currently accepted method involves use of standard polymerase chain reaction amplification of ribosomal DNA (rDNA) from the 3' 28S to 5' intergenic spacer region of the genome, and visual confirmation of amplicons of predicted size on agarose gels, after electrophoresis. This report describes development and evaluation of an automated, quantitative PCR method based upon TaqMan™ single nucleotide polymorphism (SNP) genotyping.
Standard PCR, and TaqMan SNP genotyping with newly designed primers and fluorophore-labeled probes hybridizing to sequences of complementary rDNA specific for either An. gambiae s.s. or An. arabiensis, were conducted in three experiments involving field-collected An. gambiae s.l. from western Kenya, and defined laboratory strains. DNA extraction was from a single leg, sonicated for five minutes in buffer in wells of 96-well PCR plates.
TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR. In an extensive field study, only 29 of 3,041 (0.95%) were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species), however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1%) error rate for TaqMan genotyping in mistakenly identifying species hybrids.
TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method.
Sequence detection by the 5′ nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp. The test was evaluated with 73 lung isolates and 120 tonsil isolates of A. pleuropneumoniae as well as with a collection of reference strains. By using a Ct value (cycle number in which the fluorescence exceeds the threshold defined by the software) of 30 as the cutoff limit, the 5′ nuclease assay represents a test with 100% sensitivity and 100% specificity. A high degree of reproducibility of the test was demonstrated. If samples with Ct values of ≤30 are considered positive, the detection limit of the assay was 1 CFU/reaction tube, corresponding to a 10-fold higher number of DNA templates. After cycle 30, nonspecific reactions appeared when testing dilutions of DNA templates or pure cultures of A. pleuropneumoniae, as well as when testing tonsil scrapings from specific-pathogen-free herds. The diagnostic sensitivity, as evaluated with 586 tonsil scrapings from animals infected with A. pleuropneumoniae, is the same level as that of a PCR test based on the omlA gene described previously. The 5′ nuclease assay represents a fast method for species-specific detection and identification of A. pleuropneumoniae in pure and mixed cultures. The evaluation shows, however, that a Ct value cutoff limit of ≤30 must be chosen in order to obtain reliable results. The investigation emphasizes that a thorough evaluation of the criteria used to define a positive test result is necessary.
Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at −20°C, 4°C, and room temperature was demonstrated.
Polyacrylamide gel electrophoresis of DNA fragments obtained by the polymerase chain reaction using Taq polymerase revealed the presence of multiple fragments shorter than the expected product. These abortive extension products were observed even when analysis by agarose gel electrophoresis showed only a single band. The production of prematurely terminated fragments can be exploited for the sequencing of PCR products if phosphorothioate groups are incorporated base specifically during the reaction in the presence of two oligonucleotide primers, one of which is 5'-32P-labeled. The addition of snake venom phosphodiesterase to the reaction mixture after completion of the amplification cycles digests each fragment from the 3'-end to a phosphorothioate group so that the sequence can be read by polyacrylamide gel electrophoresis.
A novel real-time quantitative polymerase chain reaction (PCR) method using an attached universal template (UT) probe is described. The UT is an approximately 20 base attachment to the 5′ end of a PCR primer, and it can hybridize with a complementary TaqMan probe. One of the advantages of this method is that different target DNA sequences can be detected employing the same UT probe, which substantially reduces the cost of real-time PCR set-up. In addition, this method could be used for simultaneous detection using a 6-carboxy-fluorescein-labeled UT probe for the target gene and a 5-hexachloro-fluorescein-labeled UT probe for the reference gene in a multiplex reaction. Moreover, the requirement of target DNA length for UT–PCR analysis is relatively flexible, and it could be as short as 56 bp in this report, suggesting the possibility of detecting target DNA from partially degraded samples. The UT–PCR system with degenerate primers could also be designed to screen homologous genes. Taken together, our results suggest that the UT–PCR technique is efficient, reliable, inexpensive and less labor-intensive for quantitative PCR analysis.
Quantification of T-cell receptor (TCR) chain families can be utilized for detection of clonal T-cell populations. Besides southern blotting and antibody-based approaches, quantitative real time PCR (qRT PCR) has been more widely applied in this context during the last years. Here, the heterogeneity of sequences within single families is the most challenging problem for exact quantification.
Vβ-families were quantified using a universal reverse primer and family-specific forward primers with TaqMan technology on a light cycler instrument. Relative concentrations were calculated considering slopes and crossing points of each PCR reaction. Total expression of α/β TCR was assessed by quantification of the constant α-chain as a further control.
The method was tested by serial dilutions of clonal T-cells in mononuclear cells from healthy volunteers. Calculated percentages were in good correspondence with qRT PCR results demonstrating high reliability. Duplicates showed excellent technical reproducibility. We analyzed blood samples of 20 healthy volunteers for determination of mean and standard deviation for each family. The method was applied both to tissue and blood samples from patients with carcinomas and hematological disorders.
We introduce a versatile method for the relative quantification of Vβ-families by real time PCR. The experimental strategy described allows the identification of alterations in the Vβ-family repertoire.
Evaluating the specificity of polymerase chain reaction (PCR) primers is an essential step in PCR primer design. The MFEprimer-2.0 server allows users to check primer specificity against genomic DNA and messenger RNA/complementary DNA sequence databases quickly and easily. MFEprimer-2.0 uses a k-mer index algorithm to accelerate the search process for primer binding sites and uses thermodynamics to evaluate binding stability between each primer and its DNA template. Several important characteristics, such as the sequence, melting temperature and size of each amplicon, either specific or non-specific, are reported on the results page. Based on these characteristics and the user-friendly output, users can readily draw conclusions about the specificity of PCR primers. Analyses for degenerate primers and multiple PCR primers are also supported in MFEprimer-2.0. In addition, the databases supported by MFEprimer-2.0 are comprehensive, and custom databases can also be supported on request. The MFEprimer-2.0 server does not require a login and is freely available at http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0. More over, the MFEprimer-2.0 command-line version and local server version are open source and can be downloaded at https://github.com/quwubin/MFEprimer/wiki/Manual/.
Scorpions are fluorogenic PCR primers with a probe element attached at the 5′-end via a PCR stopper. They are used in real-time amplicon-specific detection of PCR products in homogeneous solution. Two different formats are possible, the ‘stem–loop’ format and the ‘duplex’ format. In both cases the probing mechanism is intramolecular. We have shown that duplex Scorpions are efficient probes in real-time PCR. They give a greater fluorescent signal than stem–loop Scorpions due to the vastly increased separation between fluorophore and quencher in the active form. We have demonstrated their use in allelic discrimination at the W1282X locus of the ABCC7 gene and shown that they can be used in assays where fluorescence resonance energy transfer is required.
The action of T4 polynucleotide kinase, T4 DNA polymerase, E. coli DNA polymerase I, snake venom phosphodiesterase (VPDE) and S1 nuclease on analogues of oligothymidilates with p-s-C5' bonds and the ability of these analogues to prime the replication of poly (dA) by T4 DNA polymerase were studied. These analogues were shown to be substrates for all these enzymes. Substitution of these analogues for corresponding oligothymidilates in the reaction mixtures resulted in drop in rates of enzymic reactions. This drop in reactions rates was not significant when these oligonucleotides were phosphorylated with T4 polynucleotide kinase or used as a primers, however in comparison with oligothymidilates these analogues were found to be considerably more resistant to nucleolytic hydrolysis. Some possible applications of these analogues are discussed.
The sensitive detection of human cells in immunodeficient rodents is a prerequisite for the monitoring of micrometastasis of solid tumours, dissemination of leukaemic cells, or engraftment of haematological cells. We developed a universally applicable polymerase chain reaction method for the detection of a human-specific 850-bp fragment of the α-satellite DNA on human chromosome 17. The method allows the detection of one human cell in 106 murine cells and could be established as both, a conventional DNA polymerase chain reaction-assay for routine screening, and a quantitative real-time polymerase chain reaction-assay using TaqMan-methodology. It was applied to the following xenotransplantation systems in SCID and NOD/SCID mice: (1) In a limiting dilution assay, cells of the MDA-MB 435 breast carcinoma were injected into the mammary fat pad of NOD/SCID mice. It could be shown that 10 cells mouse−1 were sufficient to induce a positive polymerase chain reaction signal in liver and lung tissue 30 days after transplantation as an indicator for micrometastasis. At this time a palpable tumour was not yet detectable in the mammary fat pad region. (2) Cells of a newly established human acute lymphatic leukaemia were administered intraperitoneally to SCID mice. These cells apparently disseminated and were detectable as early as day 50 in the peripheral blood of living mice, while the leukaemia manifestation was delayed by day 140. (3) In a transplantation experiment using mature human lymphocytes we wanted to standardise conditions for a successful survival of these cells in NOD/SCID mice. It was established that at least 5×107 cells given intravenously were necessary and that the mice had to be conditioned by 2 Gy body irradiation to get positive polymerase chain reaction bands in several organs. (4) Engraftment studies with blood stem cells originating from cytapheresis samples of tumour patients or from cord blood were undertaken in NOD/SCID mice in order to define conditions of successful engraftment and to use this model for further optimisation strategies. The polymerase chain reaction method presented allowed a reliable prediction of positive engraftment and agreed well with the results of immunohistochemical or FACS analysis. All together, the polymerase chain reaction method developed allows a sensitive and reliable detection of low numbers of human cells in immunodeficient hosts. In combination with real-time (TaqMan) technique it allows an exact quantification of human cells. As this method can be performed with accessible material of living animals, follow up studies for the monitoring of therapeutic interventions are possible in which the survival time of mice as evaluation criteria can be omitted.
British Journal of Cancer (2002) 87, 1328–1335. doi:10.1038/sj.bjc.6600573 www.bjcancer.com
© 2002 Cancer Research UK
TaqMan PCR; xenografts; metastasis; leukaemia; haematological cells
Xenograft samples used to test anti-cancer drug efficacies and toxicities in vivo contain an unknown mix of mouse and human cells. Evaluation of drug activity can be confounded by samples containing large amounts of contaminating mouse tissue. We have developed a real-time quantitative polymerase chain reaction (qPCR) assay using TaqMan technology to quantify the amount of mouse tissue that is incorporated into human xenograft samples.
The forward and reverse primers bind to the same DNA sequence in the human and the mouse genome. Using a set of specially designed fluorescent probes provides species specificity. The linearity and sensitivity of the assay is evaluated using serial dilutions of single species and heterogeneous DNA mixtures. We examined many xenograft samples at various in vivo passages, finding a wide variety of human:mouse DNA ratios. This variation may be influenced by tumor type, number of serial passages in vivo, and even which part of the tumor was collected and used in the assay.
This novel assay provides an accurate quantitative assessment of human and mouse content in xenograft tumors. This assay can be performed on aberrantly behaving human xenografts, samples used in bioinformatics studies, and periodically for tumor tissue frequently grown by serial passage in vivo.
In the present study, we standardized a TaqMan locked nucleic acid (LNA) real-time polymerase chain reaction (PCR) probe for the accurate quantification and detection of hepatitis B virus (HBV) DNA in serum (plasma), and evaluated its methodology. LNA probe technology had a much better detection performance in HBV DNA than the common TaqMan probe. The assay based on the LNA probe had a wider linear detection range, higher sensitivity, stability and amplification efficiency, and a lower concentration of probes than the TaqMan probe. Among the 15 cases with chronic hepatitis B surface antigen (HBsAg) (+) alone, only 4 cases that were detected by TaqMan real-time PCR were negative; however, the same samples were positive by LNA real-time PCR (p<0.05). A positive correlation between viral load measurements for the 35 samples with HBV-positive DNA was detected in both LNA and TaqMan real-time PCR.
human hepatitis B virus; real-time polymerase chain reaction; probe; locked nucleic acid; TaqMan
A real-time reverse-transcription PCR was developed to detect and pathotype Newcastle disease viruses (NDV) in clinical samples. Degenerate oligonucleotide primers and TaqMan probes with nonfluorescent minor groove binder (MGB) quencher amplified and hybridized to a region in the fusion protein (F) gene that corresponds to the cleavage site of the F0 precursor, which is a key determinant of NDV pathogenicity. The application of degenerate primers and TaqMan MGB probes provided high specificity to the assay, as was shown by the successful and rapid pathotype determination of 39 NDV strains representing all the known genotypes (I to VIII) and pathotypes (lentogens/mesogens/velogens). The PCR assays specific for lentogenic and velogenic/mesogenic strains had high analytical sensitivity, detecting approximately 10 and 20 copies of the target molecule per reaction, respectively. The detection limit was also determined in terms of 50% egg infective dose (EID50) by using dilution series of virus stock solutions to be approximately 101.0 and 10−1.3 EID50/ml for lentogens and velogens/mesogens, respectively. Organ, swab, and stool specimens from experimentally infected animals were tested to prove the clinical suitability of the method. The results of this study suggest that the described real-time PCR assay has the potential to be used for the rapid detection/pathotyping of NDV isolates and qualitative/quantitative measurement of the virus load.
In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.
A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 103 CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 104 CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.
Adhesin-encoding operons (pap, sfa/foc, and afa) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. A quick and sensitive assay to identify these operons was developed by using the polymerase chain reaction (PCR). Three pairs of 25-mer primers were defined from the sequences of the DNA fragments used as probes in hybridization studies to identify each of the three operons, and the six primers were used together in a single reaction of amplification. To validate the PCR approach for detection of adhesin-encoding operons among clinical isolates, we investigated a collection of 97 E. coli isolates with the following characteristics: all isolates originated from the urine of patients with pyelonephritis, and the adhesin responsible for specific binding of the isolates to uroepithelial cells was previously characterized by phenotypic assays, as well as genotypic tests based on hybridization. There was a perfect correlation between the results obtained with the PCR approach and those previously obtained by using DNA probes. These results indicate that the PCR method, which is highly specific and easier to perform than the hybridization method, is a powerful genotypic assay for detection of adhesin-encoding operons. Thus, this assay can be recommended for clinical use to detect virulent urinary E. coli strains, as well as for epidemiological studies.
Human N-acetyltransferase 2 (NAT2) genetic polymorphism is associated with drug toxicity and/or carcinogenesis in various tissues. Knowledge of NAT2 gene structure and expression are critical for understanding these associations. Previous findings suggest that human NAT2 expression is highest in liver and gut, but expressed at functional levels in other tissues. A sensitive and specific TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) assay with intron-spanning primers was developed and used, together with a second TaqMan RT-PCR assay based on amplification of a NAT2 open reading frame (ORF) exon segment, to measure NAT2 mRNA in 29 different human tissues. Cap-dependent amplification of mRNA 5′ termini and review of public database information was done to more precisely define the NAT2 promoter(s) and to validate the quantitative RT-PCR assay design. The great majority (40/41) of NAT2 liver cDNAs had 5′ termini between 8682 and 8752 nucleotides upstream of the NAT2 ORF exon, and 34/40 5′-termini were at the -8711 and -8716 adenines. All of 59 NAT2 cDNAs with 5′ termini in this vicinity, including 40 of the liver isolates and 19 cDNAs in public databases from liver and other sources, showed direct splicing to the ORF exon, with no other non-coding exon detected. NAT2 mRNA was highest in liver, small intestine and colon and readily detected in most other tissues albeit at much lower levels. NAT2 expression in diverse human tissues provides further mechanistic support underlying associations between NAT2 genetic polymorphism, drug toxicity and/or chemical carcinogenesis.