Minor Groove binder-DNA Probe Research Articles

Real-time fluorogenic reverse transcription polymerase chain reaction assay for the specific detection of Bagaza virus

In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5'-Taq nuclease-3' minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses (Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak (n = 11), as well as samples collected from partridges during surveillance programs (n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies.

Surpassing Specificity Limits of Nucleic Acid Probes via Cooperativity

The failure to correctly identify single nucleotide polymorphisms (SNPs) significantly contributes to the misdiagnosis of infectious disease. Contrary to the strategy of creating shorter probes to improve SNP differentiation, we created larger probes that appeared to increase selectivity. Specifically, probes with enhanced melting temperature differentials (>13x improvement) to SNPs were generated by linking two probes that consist of both a capture sequence and a detection sequence; these probes act cooperatively to improve selectivity over a wider range of reaction conditions. These cooperative probe constructs (Tentacle probes) were then compared by modeling thermodynamic and hybridization characteristics to both Molecular Beacons (stem loop DNA probes) and Taqman probes (a linear oligonucleotide). The biophysical models reveal that cooperative probes compared with either Molecular beacons or Taqman probes have enhanced specificity. This was a result of increased melting temperature differentials and the concentration-independent hybridization revealed between wild-type and variant sequences. We believe these findings of order of magnitude enhanced melting temperature differentials with probes possessing concentration independence and more favorable binding kinetics have the potential to significantly improve molecular diagnostic assay functionality.

Real-Time Fluorogenic Reverse Transcription Polymerase Chain Reaction Assay for Detection of African Horse Sickness Virus

African horse sickness is an arthropod-borne disease of the equine included in the World Organization for Animal Health (OIE) list with important economic consequences for horse trade. The disease is caused by African horse sickness virus (AHSV; family Reoviridae, genus Orbivirus), which is transmitted by Culicoides midges. It is endemic in sub-Saharan Africa, spreading occasionally outside this area where the occurrence of Culicoides vectors allows virus transmission. Currently, only conventional (gel-based) reverse transcription polymerase chain reaction (RT-PCR) protocols are available for its detection; however, these methods are cumbersome and difficult to apply when large numbers of samples are to be tested, as in the case of epizootics. To overcome this problem, a real-time RT-PCR method has been developed, based on a 5'-Taq nuclease-3'-minor groove binder-DNA probe (TaqMan MGB) for detection of a wide range of AHSV serotypes and strains designed to the highly conserved region of the VP7 gene (segment 7). The method was able to detect all prototype strains from the 9 known serotypes of the virus, with a high analytical sensitivity; no cross-reactions were observed with other orbiviruses or with other viruses affecting horses. The diagnostic sensitivity was assessed using a panel of AHSV-positive tissue samples from an epizootic that occurred in Spain between 1987 and 1990. This method, which can be performed in 96-well format, is suitable for large-scale surveillance of AHSV in areas where it can potentially spread.

A New Fluorogenic Real-Time RT-PCR Assay for Detection of Lineage 1 and Lineage 2 West Nile Viruses

West Nile virus represents an emerging threat for animal and human health worldwide. This virus exhibits a marked genetic variation, with at least 2 distinct evolutionary lineages. Lineage 1 has been recognized in Africa, Asia, Europe, Oceania, and more recently in the Americas, whereas lineage 2 is restricted to Africa. Perhaps for this reason, the available real-time RT-PCR methods for detecting West Nile virus genome have mainly focused on lineage 1. However, both viruses may potentially be spread beyond their endemic areas by migratory birds. This report describes a new real-time reverse transcription-PCR (RT-PCR) method based on a 5'-Taq nuclease-3' minor groove binder DNA probe (TaqMan MGB) that allows the detection of a wide range of West Nile virus isolates, including both lineages 1 and 2. This method was able to detect West Nile viruses from different origins (North and Central Africa, Middle East, Europe, and North America), whereas other flaviviruses (Usutu, Dengue, Yellow fever) analyzed in parallel remained negative. The sensitivity achieved by this assay was 10(-2)-10(-3) pfu/tube. This method, which can be performed in 96-well format, could be suitable for the large-scale surveillance of West Nile virus in areas where both lineages can potentially spread.

Improved detection of Tritrichomonas foetus in bovine diagnostic specimens using a novel probe-based real time PCR assay

A Tritrichomonas foetus-specific 5′ Taq nuclease assay using a 3′ minor groove binder-DNA probe (TaqMan ® MGB) targeting conserved regions of the internal transcribed spacer-1 (ITS-1) was developed and compared to established diagnostic procedures. Specificity of the assay was evaluated using bovine venereal microflora and a range of related trichomonad species. Assay sensitivity was evaluated with log 10 dilutions of known numbers of cells, and compared to that for microscopy following culture (InPouch™ TF test kit) and the conventional TFR3-TFR4 PCR assay. The 5′ Taq nuclease assay detected a single cell per assay from smegma or mucus which was 2500-fold or 250-fold more sensitive than microscopy following selective culture from smegma or mucus respectively, and 500-fold more sensitive than culture followed by conventional PCR assay. The sensitivity of the conventional PCR assay was comparable to the 5′ Taq nuclease assay when testing purified DNA extracted from clinical specimens, whereas the 5′ Taq nuclease assay sensitivity improved using crude cell lysates, which were not suitable as template for the conventional PCR assay. Urine was evaluated as a diagnostic specimen providing improved and equivalent levels of T. foetus detection in spiked urine by both microscopy following culture and direct 5′ Taq nuclease detection, respectively, compared with smegma and mucus, however inconclusive results were obtained with urine samples from the field study. Diagnostic specimens ( n = 159) were collected from herds with culture positive animals and of the 14 animals positive by 5′ Taq nuclease assay, 3 were confirmed by selective culture/microscopy detection (Fisher's exact test P < 0.001). The 5′ Taq nuclease assay described here demonstrated superior sensitivity to traditional culture/microscopy and offers advantages over the application of conventional PCR for the detection of T. foetus in clinical samples.

Detection of Norovirus genogroup I and II by multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe.

BackgroundDue to an increasing number of norovirus infections in the last years rapid, specific, and sensitive diagnostic tools are needed. Reverse transcriptase-polymerase chain reactions (RT-PCR) have become the methods of choice. To minimize the working time and the risk of carryover contamination during the multi-step procedure of PCR the multiplex real-time RT-PCR for the simultaneous detection of genogroup I (GI) and II (GII) offers advantages for the handling of large amounts of clinical specimens.MethodsWe have developed and evaluated a multiplex one-tube RT-PCR using a combination of optimized GI and GII specific primers located in the junction between ORF1 and ORF2 of the norovirus genome. For the detection of GI samples, a 3'- minor groove binder-DNA probe (GI-MGB-probe) were designed and used for the multiplex real-time PCR.ResultsComparable results to those of our in-house nested PCR and monoplex real-time-PCR were only obtained using the GI specific MGB-probe. The MGB-probe forms extremely stable duplexes with single-stranded DNA targets, which enabled us to design a shorter probe (length 15 nucleotides) hybridizing to a more conserved part of the GI sequences. 97 % of 100 previously norovirus positive specimens (tested by nested PCR and/or monoplex real-time PCR) were detected by the multiplex real-time PCR. A broad dynamic range from 2 × 10^1 to 2 × 10^7 genomic equivalents per assay using plasmid DNA standards for GI and GII were obtained and viral loads between 2.5 × 10^2 and 2 × 10^12 copies per ml stool suspension were detected.ConclusionThe one-tube multiplex RT real-time PCR using a minor groove binder -DNA probe for GI is a fast, specific, sensitive and cost-effective tool for the detection of norovirus infections in both mass outbreaks and sporadic cases and may have also applications in food and environmental testing.

Interference Between D and M Types of Plum pox virus in Japanese Plum Assessed by Specific Monoclonal Antibodies and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction

ABSTRACT The dynamics of virus interference between two isolates of Plum pox virus (PPV) belonging to the main PPV types, D and M, were analyzed in Japanese plum (Prunus salicina) by challenge inoculations. To assess the consequences of a PPV-M infection on plum already infected with PPV-D, and vice versa (predominance of one of the strains, recombination, synergism, symptoms aggravation, and so on), 30 Japanese plum trees were graft inoculated with PPV-D or PPV-M isolates in quarantine conditions. One year postinoculation, in the event that the inoculated isolates were detected in the whole plant, a second challenge inoculation (PPV-M or PPV-D, respectively) was performed by grafting. The presence of PPV-D, PPV-M, or both was monitored for 7 years by double-antibody sandwich indirect enzyme-linked immunosorbent assay using specific monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) with D- and M-specific primers confirmed the serological typing. Real-time RT-PCR assays were performed using D- and M-specific fluorescent 3' minor groove binder-DNA probes, which were able to detect and quantify PPV populations in the inoculated plants with greater precision. The presence of PPV-D in Japanese plum did not cross-protect the trees against PPV-M infection. In PPV-D-infected plants, the PPV-M strain used as challenge inoculum behaved differently depending on the plum cultivar assayed. In cv. Black Diamond, PPV-M invaded the plant progressively, displacing the previous PPV-D population; whereas, in cv. Sun Gold, both PPV isolates coexisted in the plant. In contrast, the PPV-D isolate used was unable to infect plants of both cultivars in which a PPV-M population already was established. After 7 years, no synergism was observed and no recombination event between PPV-D and PPV-M genomes was detected.

Development of RT‐PCR assays using fluorogenic‐3′ minor groove binder DNA probes for detection of fruit tree viruses*

In phytosanitary certification, there is currently a need for the development of reliable, sensitive and rapid tests for the routine detection of Ilarviruses and latent viruses from fruit trees during the dormant season. We have developed real‐time RT‐PCR assays that allow the reliable detection of Prunus necrotic ringspot ilarvirus, Apple chlorotic leafspot trichovirus and Apple stem pitting virus in bark tissues of dormant wood. These assays are well adapted for the routine detection of these three viruses because they eliminate one risk of contamination by performing the whole test in a single closed tube.

Detection of single nucleotide polymorphisms in the mannose-binding lectin gene using minor groove binder-DNA probes

Structural point mutations in exon 1 at codons 52, 54 and 57 and a promotor polymorphism at −221 bp of the mannose-binding lectin (MBL) gene are associated with increased susceptibility to various infectious diseases. We developed a genotyping method based on the 5′ nuclease (TaqMan®) assay in combination with the use of minor-groove-binder (MGB) probes in screening for these mutations/polymorphisms. In contrast to conventional probes, MGB probes have a short length and can be used for detection of mutations that are in close proximity to each other, as is the case for the structural mutations in exon 1 of the MBL gene. Results obtained with the 5′ nuclease assay using MGB probes were identical with results obtained with classical techniques such as restriction fragment length polymorphism, allele-specific PCR, and sequencing. In conclusion, the 5′ nuclease assay using MGB probes is useful for large-scale screening of point mutations/polymorphisms, even when they are in close proximity.

Sensitive and specific detection of bovine immunodeficiency virus and bovine syncytial virus by 5′ Taq nuclease assays with fluorescent 3′ minor groove binder-DNA probes

Sensitive assays are required to detect bovine retroviruses in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5′ Taq nuclease assays using 3′ minor groove binder DNA probes (TaqMan ®MGB) were developed and compared to conventional PCR assays for the sensitive detection of bovine syncytial virus (BSV) and bovine immunodeficiency virus (BIV). Seven beef and dairy herds were screened to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log 10 dilutions of plasmids with inserts containing corresponding provirus sequences. Published PCR assays targeting BIV env sequences did not adequately amplify Australian BIV sequences. Pol sequences from Australian strains of BIV and BSV were used to design TaqMan ®MGB assays, which improved sensitivity 10-fold (BIV) and 100-fold (BSV), respectively, over conventional PCR tests. This is the first report of Australian sequences of BIV and BSV and the first 5′ Taq nuclease assays described to detect these viruses. These methods could be applied to future studies requiring sensitive detection of these two bovine retroviruses.

Sensitive and specific detection of proviral bovine leukemia virus by 5′ Taq nuclease PCR using a 3′ minor groove binder fluorogenic probe

Sensitive assays are required to detect proviral bovine leukemia virus (BLV) in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5′ Taq nuclease assays using 3′ minor groove binder DNA probes (TaqMan ®MGB) were developed and compared to conventional PCR assays for sensitive detection of Australian BLV. Seven beef and dairy herds were screened using DNA prepared by a variety of protocols to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log 10 dilutions of plasmids with inserted BLV sequences. Animals were also screened by the BLV standard agar-gel immunodiffusion test (AGID) and commercial enzyme linked immunosorbent assays (ELISA) for antibodies, and an ELISA for detecting viral antigens expressed (VAE) in lymphocyte cultures. The TaqMan ®MGB assay based on the pol region was the most sensitive and specific for the detection of BLV. This is the first report of a sensitive BLV 5′ Taq nuclease assay.

Optimization of a 3′-minor groove binder-DNA probe targeting the uidA gene for rapid identification of Escherichia coli O157:H7 using real-time PCR

Enterohemorrhagic Escherichia coli are harmful human pathogens capable of causing bloody diarrhea and vomiting. An important serotype commonly associated with human illness is the E. coli O157:H7 serotype. Unlike other real-time polymerase chain reaction (PCR) methods for identifying E. coli O157:H7, this study describes the development and optimization of a real-time PCR method targeting a conserved point mutation at +93 in the uidA ( gusA) gene that is unique to O157:H7, distinguishing it from non-O157:H7 serotypes. A TET-labeled Minor Groove Binder (MGB) DNA probe was designed for use in a 5′ nuclease PCR assay. Using a panel of two E. coli O157:H7 strains, three E. coli non-O157:H7 strains, and one non- E. coli species, the assay was optimized for the specific detection of the E. coli O157:H7 strains. Optimal conditions were identified at high anneal/extend temperatures, low magnesium concentrations, and low probe concentrations, resulting in correct identification of E. coli O157:H7 and non-O157:H7 strains. The improved specificity of MGB probes for single base pair mismatches such as the +93 uidA mutation provides a novel approach towards rapid identification of E. coli O157:H7.

Development of Real-Time PCR for the Rapid Detection of Episomal Banana streak virus (BSV).

A real-time assay for the detection of episomal Banana streak virus (BSV; strain OL) in banana and plantains that carry integrated BSV sequences is described. Primers specific to the viral DNA were designed using the viral sequence integrated into the cv. Obino l'Ewai genome and the sequence of the genomic DNA of the infecting virus strain OL. They amplify a sequence of 1,336 bp that is detected in real-time by a short fluorogenic 3' minor groove binder DNA probe. This method enables reproducible and specific detection of episomal BSV from purified DNA as well as from crude extracts from infected plants. The assay is rapid, adaptable for large-scale experiments, and circumvents carryover problems.

Detection of apple chlorotic leaf spot virus using a 5′ nuclease assay with a fluorescent 3′ minor groove binder-DNA probe

The development of a real-time 5′ nuclease RT-PCR assay for the detection of apple chlorotic leaf spot virus (ACLSV) from infected plant material is described. A short fluorogenic 3′ minor groove binder-DNA hydrolysis probe was used to circumvent genome variability between isolates and target a short conserved sequence. The covalent attachment of the minor groove binder moiety at the 3′ end of the probe increased the probe/target duplex stability and raised the melting temperature to a range suitable for real-time analysis. The method is rapid, sensitive and takes place within a single tube without post-PCR handling of the amplification products.

Rapid and Homogenous Detection of Apple stem pitting Virus by RT-PCR and a Fluorogenic 3' Minor Groove Binder-DNA Probe

Real-time PCR with fluorogenic hydrolysis probes (5' nuclease assay) is increasingly used for the detection of pathogens for diagnostic purposes. Nevertheless, the size of the probes, usually 25–40 nucleotides, might limit their use to detect pathogens with high genome variability between isolates, where an identical sequence cannot be found without multiple mismatches. In this report, we describe a 5' nuclease assay, to detect Apple stem pitting virus, based on the use of a shorter probe which is chemically modified with a minor groove binder in order to increase duplex stability and raise the melting temperature to a value suitable for real-time analysis. The short size of the probe, which is critical to target a conserved cluster sequence of 14 nucleotides in the RNA polymerase gene, circumvents the genome variability of the virus. The assay correlates at 96 percent with gel analysis and is more reliable than biological indexing to detect Apple stem pitting virus field isolates. It is fast and fully compatible with automation, and therefore particularly suitable for plant certification.