Real-Time PCR Detection of Brucella abortus: a Comparative Study of SYBR Green I, 5′-Exonuclease, and Hybridization Probe Assays

Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5′-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.     

Universal DNA array detection of small insertions and deletions in BRCA1 and BRCA2

Array-based mutation detection methodology typically relies on direct hybridization of the fluorescently labeled query sequence to surface-bound oligonucleotide probes. These probes contain either small sequence variations or perfect-match sequence. The intensity of fluorescence bound to each oligonucleotide probe is intended to reveal which sequence is perfectly complementary to the query sequence. However, these approaches have not always been successful, especially for detection of small frameshift mutations. Here we describe a multiplex assay to detect small insertions and deletions by using a modified PCR to evenly amplify each amplicon (PCR/PCR), followed by ligase detection reaction (LDR). Mutations were identified by screening reaction products with a universal DNA microarray, which uncouples mutation detection from array hybridization and provides for high sensitivity. Using the three BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population (BRCA1 185delAG; BRCA1 5382insC; BRCA2 6174delT) as a model system, the assay readily detected these mutations in multiplexed reactions. Our results demonstrate that universal microarray analysis of PCR/PCR/LDR products permits rapid identification of small insertion and deletion mutations in the context of both clinical diagnosis and population studies.     

Different reactions obtained using the same DNA detection reagents for Thai and Korean hepatopancreatic parvovirus of penaeid shrimp.

Hepatopancreatic parvovirus (HPV) can cause stunted growth and death in penaeid shrimp including Penaeus monodon. We used PCR primers and a commercial DNA probe designed from HPV of Penaeus chinensis (HPVchin) to examine HPV-infected Thai P. monodon (HPVmon). We found that the PCR primers produced a 732 bp DNA amplicon rather than the 350 bp amplicon obtained with HPVchin template and that the DNA probe gave weak to variable in situ DNA hybridization results. In addition, hybridization to PCR products from HPVmon was weak compared with hybridization with PCR products from HPVchin. By contrast, the 732 bp amplicon hybridized strongly with HPVmon-infected cells by in situ hybridization but not with uninfected shrimp tissue or other shrimp viruses, thus confirming its origin from HPVmon. Cloning, sequencing and analysis of the 732 bp amplicon showed that 696 bp (excluding the primer sequences) contained 47% GC content and had only 78% homology to 701 aligned bases from a 3350 bp DNA fragment of HPVchin from GenBank. These results explain why the reagents based on HPVchin gave a different PCR product and weak hybridization results with HPVmon, and they show that multiple primers or degenerate primers may be necessary for general detection of HPV varieties. Together with previously published information on the estimated total genome sizes for HPVchin (approximately 4 kb) and HPVmon (approximately 6 kb), these data support the contention that HPVchin and HPVmon are different varieties or species, in spite of their similar histopathology.     

Detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays

Polymerase chain reaction (PCR) assays have proven useful for detection of rodent parvoviruses in animals and contaminated biological materials. Fluorogenic nuclease PCR assays combine PCR with an internal fluorogenic hybridization probe, eliminating post-PCR processing and potentially enhancing specificity. Consequently, three fluorogenic nuclease PCR assays were developed, one that detects all rodent parvoviruses, one that specifically detects minute virus of mice (MVM), and one that specifically detects mouse parvovirus 1 (MPV) and hamster parvovirus (HaPV). When rodent parvoviruses and other rodent DNA viruses were evaluated, the rodent parvovirus assay detected only rodent parvovirus isolates, whereas the MVM and MPV/HaPV assays detected only the MVM or MPV/ HaPV isolates, respectively. Each assay detected the equivalent of 10 or fewer copies of target template, and all fluorogenic nuclease PCR assays exceeded the sensitivities associated with previously reported PCR assays and mouse antibody production testing. In addition, each fluorogenic nuclease PCR assay detected the targeted parvovirus DNA in tissues obtained from mice experimentally infected with MVM or MPV. Results of these studies indicate that fluorogenic nuclease PCR assays provide a potentially high-throughput, PCR-based method to detect rodent parvoviruses in infected mice and contaminated biological materials.     

Comparison of three different PCR methods for detection of Brucella spp in human blood samples

In the diagnosis of human brucellosis, PCR could be a more sensitive technique than blood cultures and more specific than conventional serological tests. We compared three different PCR methods for the detection of Brucella spp. and we studied whether human genomic DNA affect the sensitivity of three primer pairs for the detection of Brucella DNA in a peripheral-blood PCR assay. These three pairs of primers amplified three different fragments included in: (i). a gene encoding a 31-kDa Brucella abortus antigen (primers B4/B5), (ii). a sequence 16S rRNA of B. abortus (primers F4/R2), and (iii). a gene encoding an outer membrane protein (omp-2) (primers JPF/JPR). The three primers assayed showed a difference in sensitivity for detecting purified Brucella DNA, ranging between 8 fg and 20 pg. However, the sensitivity of the primers F4/R2 and B4/B5 was affected by the presence of human DNA while the primers JPF/JPR were not. Therefore, although the sensitivity of PCR using primers F4/R2 is affected by human DNA, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis.     

Effects of target length on the hybridization efficiency and specificity of rRNA-based oligonucleotide microarrays

The effect of target size on microarray hybridization efficiencies and specificity was investigated using a set of 166 oligonucleotide probes targeting the 16S rRNA gene of Escherichia coli. The targets included unfragmented native rRNA, fragmented rRNA ( approximately 20 to 100 bp), PCR amplicons (93 to 1,480 bp), and three synthetic single-stranded DNA oligonucleotides (45 to 56 bp). Fluorescence intensities of probes hybridized with targets were categorized into classes I (81 to 100% relative to the control probe), II (61 to 80%), III (41 to 60%), IV (21 to 40%), V (6 to 20%), and VI (0 to 5%). Good hybridization efficiency was defined for those probes conferring intensities in classes I to IV; those in classes V and VI were regarded as weak and false-negative signals, respectively. Using unfragmented native rRNA, 13.9% of the probes had fluorescence intensities in classes I to IV, whereas the majority (57.8%) exhibited false-negative signals. Similar trends were observed for the 1,480-bp PCR amplicon (6.6% of the probes were in classes I to IV). In contrast, after hybridization of fragmented rRNA, the percentage of probes in classes I to IV rose to 83.1%. Likewise, when DNA target sizes were reduced from 1,480 bp to 45 bp, this percentage increased approximately 14-fold. Overall, microarray hybridization efficiencies and specificity were improved with fragmented rRNA (20 to 100 bp), short PCR amplicons (<150 bp), and synthetic targets (45 to 56 bp). Such an understanding is important to the application of DNA microarray technology in microbial community studies.     

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.     

Solid-phase time-resolved fluorescence detection of human immunodeficiency virus polymerase chain reaction amplification products.

A new assay system for the detection of polymerase chain reaction (PCR) amplification products is presented. This single-pot sandwich assay system employs solid-support oligonucleotide-coated capture beads, a rare earth metal chelate-labeled probe, and a time-resolved fluorescence detection. The new assay system was evaluated for various reaction conditions including, DNA denaturation time, hybridization salt concentration, probe concentration, and hybridization time, all of which are important in designing an assay with a high level of sensitivity for the detection of duplex DNA. This nonisotopic assay system was applied to the detection of purified human immunodeficiency virus (HIV) DNA and sensitivity was compared with agarose gel electrophoresis and slot blot hybridization using a 32P-labeled probe. We were able to detect the amplified product from one copy of HIV DNA after 35 cycles of PCR amplification in less than 30 min using this assay, which compared with one copy by gel electrophoresis after 40 cycles of PCR amplification and one copy by slot blot hybridization after 35 cycles of PCR amplification and an overnight exposure of the autoradiogram. Thus, this assay is rapid, sensitive, and easy to use.     

Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella

Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We developed a real-time PCR assay to detect the presence of Salmonella species using these fluorogenic reporter molecules. A 122-base-pair section of the himA was used as the amplification target. Molecular beacons were designed to recognize a 16-base-pair region on the amplicon. As few as 2 colony-forming unit (CFU) per PCR reaction could be detected. We also demonstrated the ability of the molecular beacons to discriminate between amplicons obtained from similar species such as Escherichia coli and Citrobacter freundii in real-time PCR assays. These assays could be carried out entirely in sealed PCR tubes, enabling fast and direct detection of Salmonella in a semiautomated format.     

Improved assay-dependent searching of nucleic acid sequence databases

Nucleic acid-based biochemical assays are crucial to modern biology. Key applications, such as detection of bacterial, viral and fungal pathogens, require detailed knowledge of assay sensitivity and specificity to obtain reliable results. Improved methods to predict assay performance are needed for exploiting the exponentially growing amount of DNA sequence data and for reducing the experimental effort required to develop robust detection assays. Toward this goal, we present an algorithm for the calculation of sequence similarity based on DNA thermodynamics. In our approach, search queries consist of one to three oligonucleotide sequences representing either a hybridization probe, a pair of Padlock probes or a pair of PCR primers with an optional TaqMantrade mark probe (i.e. in silico or 'virtual' PCR). Matches are reported if the query and target satisfy both the thermodynamics of the assay (binding at a specified hybridization temperature and/or change in free energy) and the relevant biological constraints (assay sequences binding to the correct target duplex strands in the required orientations). The sensitivity and specificity of our method is evaluated by comparing predicted to known sequence tagged sites in the human genome. Free energy is shown to be a more sensitive and specific match criterion than hybridization temperature.     

A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus

PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50–80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified mixture in broad-range PCR applications.     

Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue

As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and “amplifiability” for aCGH and other downstream applications. Phenol–chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues.     

Evaluation of culture, ELISA and PCR assays for the detection of Salmonella in seafood

Aims: The study evaluated the efficiency of culture, enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) assays for the detection of Salmonella in naturally contaminated seafood. Methods and Results: In this study, 215 seafood samples comprising fish, shrimp, crab, clam, mussel, oyster, squid, cuttlefish and octopus from fish market of Cochin (India), were compared by culture, ELISA and PCR methods. Bacteriological Analytical Manual (BAM), U.S. Food and Drug Administration (USFDA) method was followed for culture assay, and Salmonella Tek, a commercial sandwich ELISA kit, was used for ELISA assay. Salmonella‐specific PCR assay was developed for 284 bp Salmonella‐specific invA gene amplicon. PCR assay exhibited 31·6% seafood positive for Salmonella followed by ELISA (23·7%) and culture method (21·3%). There was fair to excellent agreement between culture, ELISA and PCR assays (kappa coefficient values ranging from 0·385 to 1·0) for different seafood samples. Conclusion: The investigation revealed the greater concordance between culture and ELISA methods for seafood. Among the three methods, PCR assay was most sensitive. Lower detection rate with culture and ELISA assays could be attributed to greater sensitivity of the PCR method in the detection of Salmonella in seafood. Significance and Impact of the Study: We propose the incorporation of dual tests based on different principle and procedure for the routine analysis of Salmonella in seafood.     

Occurrence and linkage of genes coding for resistance to sulfonamides, streptomycin and chloramphenicol in bacteria of the genera Pasteurella and Mannheimia

Twenty-three isolates of the two genera Pasteurella (P.) and Mannheimia (M.) were analysed for the presence of genes specifying resistance to sulfonamides, streptomycin, and chloramphenicol. Specific PCR assays for the detection of the genes sulII, strA and catAIII, but also for the confirmation of their physical linkage were developed. A resistance gene cluster consisting of all three genes and characterised by a PCR amplicon of 2.2 kb was detected on four different types of plasmids and also in the chromosomal DNA of seven isolates. Physically linked sulII and strA genes were detected on three different types of plasmids and in the chromosomal DNA of three isolates. Sequence analysis of the different PCR amplicons revealed that these genes were present in either the orientation sulII-strA separated by differently sized spacer sequences, or strA-sulII. A truncated strA gene preceding a sulII gene was also detected in two cases.     

Detection of legionellae in hospital water samples by quantitative real-time LightCycler PCR

Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionella spp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed of Legionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44 Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r = 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitative L. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developed Legionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.     

Detection of the Hereditary Hemochromatosis Gene Mutation by Real-Time Fluorescence Polymerase Chain Reaction and Peptide Nucleic Acid Clamping

Hereditary hemochromatosis (HH), an iron overload disease, is the most common known inheritable disease. The most prevalent form of HH is believed to be the result of a single base-pair mutation. We describe a rapid homogeneous mutation analysis method that does not require post-polymerase chain reaction (PCR) manipulations. This method is a marriage of three emerging technologies: rapid cycling PCR thermal cyclers, peptide nucleic acid (PNA) probes, and a new double-stranded DNA-selective fluorescent dye, Sybr Green I. The LightCycler is a rapid thermal cycler that fluorometrically monitors real-time formation of amplicon with Sybr Green I. PNAs are DNA mimics that are more sensitive to mismatches than DNA probes, and will not serve as primers for DNA polymerases. PNA probes were designed to compete with PCR primers hybridizing to the HH mutation site. Fully complemented PNA probes at an 18:1 ratio over DNA primers with a mismatch result in suppression of amplicon formation. Conversely, PNA probes with a mismatch will not impair the binding of a complementary primer, culminating in amplicon formation. A LightCycler-based rapid genetic assay has been developed to distinguish HH patients from HH carriers and normal individuals using PNA clamping technology.     

Design considerations for array CGH to oligonucleotide arrays.

BACKGROUND: Representational oligonucleotide microarray analysis has been developed for detection of single nucleotide polymorphisms and/or for genome copy number changes. In this process, the intensity of hybridization to oligonucleotides arrays is increased by hybridizing a polymerase chain reaction (PCR)-amplified representation of reduced genomic complexity. However, hybridization to some oligonucleotides is not sufficiently high to allow precise analysis of that portion of the genome. METHODS: In an effort to identify aspects of oligonucleotide hybridization affecting signal intensity, we explored the importance of the PCR product strand to which each oligonucleotide is homologous and the sequence of the array oligonucleotides. We accomplished this by hybridizing multiple PCR-amplified products to oligonucleotide arrays carrying two sense and two antisense 50-mer oligonucleotides for each PCR amplicon. RESULTS: In some cases, hybridization intensity depended more strongly on the PCR amplicon strand (i.e., sense vs. antisense) than on the detection oligonucleotide sequence. In other cases, the oligonucleotide sequence seemed to dominate. CONCLUSION: Oligonucleotide arrays for analysis of DNA copy number or for single nucleotide polymorphism content should be designed to carry probes to sense and antisense strands of each PCR amplicon to ensure sufficient hybridization and signal intensity.     

A fluorescence polarization assay using oligonucleotide probes for the rapid detection of verotoxin-producing Escherichia coli

A hybridization assay using fluorescence polarization was combined with the asymmetric polymerase chain reaction (PCR) in a method for the detection of the verotoxin type 2 gene of verotoxin-producing Escherichia coli. Six oligonucleotide probes labeled with FITC were designed and evaluated. One of these gave a detection limit of 10(3) colony forming units per assay, and assay results could be obtained within 5 min after PCR. It appears that the detection limit was restricted mainly by the extent and fidelity of PCR amplification, rather than by the sensitivity of the fluorescence polarization technique, indicating that good probe design facilitates the rapid detection of the PCR product. The fluorescence polarization assay, in conjunction with DNA amplification by PCR, is a powerful and widely applicable method for the rapid and sensitive detection of oligonucleotide sequences.     

Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes

Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.     

Quantitation of polymerase chain reaction products by hybridization-based assays with fluorescent, colorimetric, or chemiluminescent detection.

In this report two nonradioactive assays for quantitative analysis of polymerase chain reaction (PCR) products are presented. In the first assay, magnetic beads coated with streptavidin were used to capture biotinylated PCR fragments. After hybridization with a hapten-labeled probe, these beads were analyzed either by flow cytometry (method A) or by immunoenzymatic reactions (method B). Using a dilution series of purified PCR products, we consistently found a lower detection limit of 1.5 fmol for method A than the 0.15-fmol limit for method B. In the second assay we used the peroxidase-based enhanced chemiluminescence system in combination with a cooled charge-coupled device camera to quantify PCR fragments that were spotted on membranes. A linear logarithmic response was observed between the amount of light produced within a certain time interval and the number of DNA molecules. With an exposure time of 5 min, a detection limit of 0.15 fmol was found. Longer exposure times did not result in a higher sensitivity. We conclude that the assays are of sufficient sensitivity for application in quantitative PCR strategies. The nonradioactive technology facilitates implementation of these assays in routine settings.