TaqMan Minor Groove Binding Probes Research Articles

A quantitative reverse transcription-polymerase chain reaction for detection of Getah virus

Getah virus (GETV), a mosquito-borne alphavirus, is an emerging animal pathogen causing outbreaks among racehorses and pigs. Early detection of the GETV infection is essential for timely implementation of disease prevention and control interventions. Thus, a rapid and accurate nucleic acid detection method for GETV is highly needed. Here, two TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were developed. The qRT-PCR primers and TaqMan MGB probe were designed based on the conserved region of nsP1 and nsP2 genes of 23 GETV genome sequences retrieved from GenBank. Only the qRT-PCR assay using nsP2-specific primers and probe detected all two Malaysia GETV strains (MM2021 and B254) without cross-reacting with other closely related arboviruses. The qRT-PCR assay detected as few as 10 copies of GETV RNA, but its detection limit at the 95% probability level was 63.25 GETV genome copies (probit analysis, P ≤ 0.05). Further validation of the qRT-PCR assay using 16 spiked simulated clinical specimens showed 100% for both sensitivity and specificity. In conclusion, the qRT-PCR assay developed in this study is useful for rapid, sensitive and specific detection and quantification of GETV.

Single-tube multiplex real-time PCR assay for rapid and reliable detection of HLA-A*31:01 allele.

HLA-A*31:01 has been associated with carbamazepine-induced hypersensitivity reactions. HLA-A*31:01 genetic testing is recommended before the initiation of carbamazepine therapy. A novel real-time PCR assay was designed for HLA-A*31:01 detection by allele-specific primers and TaqMan minor groove binding probes. The genotyping results in 100 subjects by the established method who were in 100% agreement with the sequencing-based typing method. The assay presents a sensitivity of 1 (95% CI: 0.69-1.00), a specificity of 1 (95% CI: 0.96-1.00) and a positive and negative predictive value of 1. The carrier rates of HLA-A*31:01 in Tibetan (n=45), Han Chinese (n=100), Miaos (n=48) and Khalkhas (n=48) were 22.2, 10, 4.2 and 18.8%, respectively. This assay is reliable to detect HLA-A*31:01 and would be useful to prevent carbamazepine-induced hypersensitivity reactions.

Atypical Chemokine Receptor 1 Polymorphism can not Affect Susceptibility to Hepatitis C Virus.

Background:Hepatitis C virus has infected 130 to 150 million individuals globally. Atypical chemokine receptor 1 has become a focus of research because of its diverse roles in different diseases. However, little is known regarding the association of atypical chemokine receptor 1 polymorphism with susceptibility to hepatitis C virus.Aims: To determine the association of an atypical chemokine receptor 1 polymorphism (rs12075) with hepatitis C virus susceptibility.Study Design: Case-control study.Methods: We collected blood samples from 231 patients infected with hepatitis C virus and 239 blood donors as control subjects. Genotyping of atypical chemokine receptor 1 was performed using a 5ˊ-nuclease assay with TaqMan-minor groove binding probes. Comparisons between hepatitis C virus-infected patients and control subjects were assessed using Fisher’s exact test.Results: The genotype frequencies of FY*A/FY*A, FY*A/FY*B and FY*B/FY*B were 86.1%, 13.9% and 0% in the patient group, and 86.2%, 13.4% and 0.4% in the control group, respectively. The difference in atypical chemokine receptor 1 genotype frequencies between hepatitis C virus-infected patients and control group was not significant (p=1.00, OR=1.004, 95% CI=0.594-1.695). FY*A and FY*B allele frequencies were 93.1% and 6.9% in the patient group, and 92.9% and 7.1% in the control group, respectively. The difference in atypical chemokine receptor 1 allele frequencies between hepatitis C virus-infected patients and the control group was not significant (p=1.00, OR=0.972, 95% CI=0.589-1.603).Conclusion: Our result indicates that atypical chemokine receptor 1 polymorphism (rs12075) does not affect susceptibility to hepatitis C virus.

A New Method for Analyzing the Duffy Blood Group Genotype by TaqMan Minor Groove Binding Probes.

Duffy blood group genotyping is useful to ensure transfusion safety and determine the association of Duffy blood group polymorphism with diseases, and therefore has its clinical significance. In order to improve the existing methods for genotyping of Duffy blood group, which normally require post-PCR manipulation, a new method was developed by using 5'-nuclease assay (NA) with TaqMan minor groove binding (MGB) probes. Primers and TaqMan-MGB probes were designed and synthesized to genotype FY*A and FY*B alleles at Duffy blood group locus on a real-time PCR platform. A total of 120 samples were genotyped by using the new 5'-NA and conventional polymerase chain reaction with allele-specific primers (PCR-ASP). The results obtained by the two methods were compared. There was a complete concordance of results for all samples genotyped by 5'-NA and PCR-ASP. The retesting results of 5'-NA were consistent with those of the initial testing. The detection limit of 5'-NA was determined as 100 pg per reaction. The FY*A and FY*B allelic frequencies were 93.3% and 6.7% respectively in the Chinese Han population in Dalian. The 5'-NA for genotyping of Duffy blood group is simple, rapid, reliable, reproducible, sensitive, and high-throughput and is superior to PCR-ASP used in routine genotyping.

Detection and quantification by PCR assay of the biocontrol agent Pantoea agglomerans CPA-2 on apples

The registration of biological control agents requires the development of monitoring systems to detect and quantify the agent in the environment. Pantoea agglomerans CPA-2 is an effective biocontrol agent for postharvest diseases of citrus and pome fruits. The monitoring of CPA-2 in postharvest semi-commercial trials was evaluated by Rodac impression plates and the colonies isolated were confirmed by conventional PCR using the SCAR primers PAGA1 and PAGB1. Samples were taken from different surfaces that had contact with CPA-2, the surrounding environment and working clothes worn by handlers. Moreover, population dynamics of the strain CPA-2 were determined on apple surfaces using both the classical plating technique and real-time quantitative PCR (qPCR). A qPCR assay using a 3′-minor groove-binding (MGB) probe was developed for the specific detection and quantification of P. agglomerans strain CPA-2. Based on the nucleotide sequence of a SCAR fragment of CPA-2, one primer set and TaqMan MGB probe were designed. The primers SP2-F/SP2-R and the TaqMan MGB probe showed a specific detection of strain CPA-2 on apple surfaces, which was verified tested against purified DNA from 17 strains of P. agglomerans, 4 related Pantoea species, and 21 bacterial strains from other genera isolated from whole and also freshly-cut fruit and vegetables. The detection level was approximately 103 cells per reaction, and the standard curve was linear within a range of 5log units. Results from semi-commercial trials showed that CPA-2 had a low impact. The maximum persistence of P. agglomerans CPA-2 was not longer than 5days in plastic boxes stored at 0°C. Significant differences in CPA-2 population level dynamics were observed in results obtained by qPCR and dilution plating. These differences may indicate the presence of non-degraded DNA from non-viable cells. In conclusion, qPCR is a novel potential tool to quickly and specifically monitor recent surface colonisation by CPA-2 populations on apple surfaces during large-scale experiments that could ensure efficient and successful treatments.

Molecular discrimination of toxic and non-toxic Alexandrium species (Dinophyta) in natural phytoplankton assemblages from the Scottish coast of the North Sea

Molecular methods provide promising tools for routine detection and quantification of toxic microalgae in plankton samples. To this end, novel TaqMan minor groove binding probes and primers targeting the small (SSU) or large (LSU) ribosomal subunit (rRNA) were developed for two species of the marine dinoflagellate genus Alexandrium (A. minutum, A. tamutum) and for three groups/ribotypes of the A. tamarense species complex: Group I/North American (NA), Group II/Mediterranean (ME) and Group III/Western European (WE). Primers and probes for real-time quantitative PCR (qPCR) were species-specific and highly efficient when tested in qPCR assays for cross-validation with pure DNA from cultured Alexandrium strains. Suitability of the qPCR assays as molecular tools for the detection and estimation of relative cell abundances of Alexandrium species and groups was evaluated from samples of natural plankton assemblages along the Scottish east coast. The results were compared with inverted microscope cell counts (Utermöhl technique) of Alexandrium spp. and associated paralytic shellfish poisoning (PSP) toxin concentrations. The qPCR assays indicated that A. tamarense (Group I) and A. tamutum were the most abundant Alexandrium taxa and both were highly positively correlated with PSP toxin content of plankton samples. Cells of A. tamarense (Group III) were present at nearly all stations but in low abundance. Alexandrium minutum and A. tamarense (Group II) cells were not detected in any of the samples, thereby arguing for their absence from the specific North Sea region, at least at the time of the survey. The sympatric occurrence of A. tamarense Group I and Group III gives further support to the hypothesis that the groups/ribotypes of the A. tamarense species complex are cryptic species rather than variants belonging to the same species.

Influence of interferon-α treatment outcome in polycythemia vera and essential thrombocythemia by genetic polymorphism in IL28B

Background: Interferon-α (IFN-α) is a therapeutic option in high risk patients with Myeloproliferative Neoplasms (MPNs). The response to IFN-α treatment of chronic hepatitis C was shown to be strongly influenced by several related single nucleotide polymorphisms (SNP) in a region adjacent to the IL28B gene. The objective of this study was to investigate the possibility of similar influence of IL28B gene polymorphism in IFN-α treated MPN patients. Methods: 20 patients with polycythemia vera (PV) or essential thrombocythemia (ET) treated with IFN-α included, 7 were earlier treated with hydroxyurea. Hematologic response was evaluated using the European Leukemia Net (ELN) criteria. DNA from whole blood samples was isolated using MagNA Pure LC DNA Isolation Kit I for IL28B genotyping, and the variability at rs12979860, rs12980275 and rs8099917 were determined by allelic discrimination assays using Taqman minor groove binding probes. Results: The IFN-α treatment reduced platelet count ( p <0.05), and in patients with polycythemia vera (PV) also hemoglobin, hematocrit and white blood cell counts declined ( p <0.05). Hematologic response by ELN criteria was significantly influenced by the IL28B SNP. Among 20 patients with PV or ET, the CC genotype at rs12979860 was found in 8/11 patients (73%) with hematologic CR as compared with 1/9 (11%) with PR or NR ( p =0.010). Conclusion: The results in this pilot study indicate that variation in IL28B genotype might influence hematologic response in IFN-α treated MPN patients.

Development of a real‐time PCR assay for Pseudomonas cichorii, the causal agent of midrib rot in greenhouse‐grown lettuce, and its detection in irrigating water

Midrib rot is an emerging disease in greenhouse production of lettuce caused by Pseudomonas cichorii, and probably introduced through contaminated irrigation water. Concentrations of 100 CFU mL−1 are enough to induce the typical midrib rot symptoms. A sensitive real‐time PCR assay was developed, based on a 90‐bp amplicon from the pathogenicity gene cluster hrcRST and a Taqman Minor Groove Binding probe. Specificity of the assay was tested with 39 P. cichorii strains, including the type strain, and 89 strains from 83 other Pseudomonas species. The relationship between detection signals and P. cichorii DNA concentrations was linear over 6‐logs. Detection threshold with excellent reproducibility was 500 fg of DNA or about 70 genome copies. Sample preparation and DNA isolation were optimized to allow detection in 1 L water samples. The assay was first evaluated with greenhouse irrigation water spiked with serial dilutions of P. cichorii. The calculated cell numbers obtained with real‐time PCR were 10‐fold lower than plate counts of actual spiked cells. However, the assay consistently detected 100 CFU per reaction, corresponding to the detection of 1 CFU mL−1 of irrigation water, which is well below the concentration needed for midrib rot infection. Finally, the assay proved to be valuable for detecting infective P. cichorii concentrations in the irrigation water of a commercial lettuce production greenhouse.

A species‐specific real‐time PCR assay for identification of three lichen‐forming fungi, Lobaria pulmonaria, Lobaria immixta and Lobaria macaronesica

We present a multiplex real-time PCR assay for the simultaneous identification of three morphologically similar species of lichen-forming fungi, Lobaria pulmonaria, Lobaria immixta and Lobaria macaronesica. Based on TaqMan MGB (minor groove binding) probes targeting the fungal internal transcribed spacer (ITS nrDNA) region, our assay unambiguously identifies known samples from all the three species, thus providing a powerful alternative to the more expensive DNA-sequencing techniques.

Inositol hexaphosphate alters mucin expression in the intestinal mucosa of mice

Inositol hexaphosphate (IP6 or phytic acid) is thought to be the active ingredient in legumes and cereals that provides anti‐cancer activity. Our goal is to understand the influence of IP6 on mucin expression (Muc1 and Muc2, specifically) in the mouse intestine. C57BL/6 mice were randomized into 3 groups of 6 mice. Mice were orally dosed with IP6 (0, 1, 2 g/kg BW) for 5 days and sacrificed on the sixth day. Sections of the small intestine were removed, fixed in methacarn, sectioned and stained with Alcian blue‐PAS to visualize mucins. Mucosa from the jejunum and colon were removed and total RNA prepared using Trizol®. cDNA was prepared using random hexamers and TaqMan minor groove binding probes were used for real time PCR amplification of message for Muc1 and Muc2. Expression of Muc1 in the jejunum was minimal whereas that of Muc2 increased 1.4‐fold at the higher IP6 dose rate. In the colon, expression of both Muc1 and Muc2 increased in mice dosed with IP6. Expression of Muc1 increased 1.6‐fold and 3.8‐fold in mice dosed with 1 and 2 g/kg IP6, respectively. Expression of Muc2 increased 2.0‐fold and 1.7‐fold in mice dosed with 1 and 2 g/kg IP6, respectively. Alcian blue‐PAS stained sections of the colon showed an increase in mucus. Thus, IP6 seems to increase the expression of Muc1 and Muc2 in the mucosa lining of the small intestine, particularly in the colon.Research was supported by East Tennessee State University and Mayo Clinic.