TaqMan Minor Groove Binder Probes Research Articles

Development of a TaqMan MGB RT-PCR for the rapid detection of H3 subtype avian influenza virus circulating in China

Previous studies demonstrated that the H3 avian influenza virus (AIV) in China is isolated most frequently from wild birds and live poultry markets. However, there is no subtype-specific real-time polymerase chain reaction (RT-PCR) available for the rapid and highly sensitive identification of H3 AIV. In this study, a TaqMan minor groove binder (MGB) probe and a pair of primers were designed based on a conserved region in the hemagglutinin gene of H3 AIV. These were used to generate an H3-MGB RT-PCR assay that recognizes only H3 AIV. The detection limit of the H3-MGB RT-PCR was 10 copies of DNA per reaction when 10-fold serial dilutions of T-H3HA plasmid were used as the template. This was 1000-times more sensitive than conventional RT-PCR. In experimental samples obtained from oropharyngeal swabs or cloacal swabs, the virus was detected in all ducks using H3-MGB RT-PCR, whereas only one duck tested positive for the virus in oropharyngeal swabs tested using conventional RT-PCR. The H3-MGB RT-PCR assay developed in this study is a sensitive and rapid tool for screening H3 AIV in China.

Quantitation of substitutions at amino acid 70 in hepatitis C virus genotype 1b.

BackgroundSubstitutions of amino acid (aa) 70 in the core region of hepatitis C virus genotype 1b (HCV 1b) are a predictor of the non-virological response to pegylated interferon plus ribavirin (PEG-IFN/RBV) therapy. The aim of our study was to develop quantitative real-time reverse transcription polymerase chain reaction (qPCR) assays to quantify wild-type (70 W) and mutant (70 M) strains of HCV 1b.MethodsWe used the TaqMan system to quantify strains 70 W and 70 M. Codon 70 in the HCV 1b core region can be either CGN or CAN, therefore degenerate TaqMan minor groove binder (MGB) probes with inosine were used. We determined detection limits, sensitivity and specificity of the methods developed. Direct sequencing and cloning of the HCV core region were used to confirm the reliability of our new system. Serum samples from 138 Chinese patients infected with HCV 1b were examined with the system we developed and compared with results obtained from the Roche TaqMan RT-PCR HCV RNA quantitation system.ResultsDegenerate MGB probes were able to clearly distinguish 70 W from 70 M. The detection limit was 103 copies/mL. Cross-reactivity tests confirmed the specificity of our method. Our system can effectively quantify 70 W and 70 M for 99.6% of patients with HCV 1b. Further tests involving cloning and sequencing confirmed the reliability of our system.ConclusionsWe developed an assay system using degenerate TaqMan MGB probes with inosine to quantify wild-type and mutant viral RNAs of the HCV 1b core region at aa 70. Our developed assay system had high levels of sensitivity and accuracy, and could prove useful in investigating dynamic changes during PEG-IFN/RBV therapy to assess virological responses.

Identification of Campylobacter jejuni and determination of point mutations associated with macrolide resistance using a multiplex TaqMan MGB real-time PCR

Campylobacter jejuni, one of the most important food-borne pathogens, develops resistance to macrolides by acquiring point mutations in the 23S rDNA gene (e.g. A2075G or A2074C) and in ribosomal protein L4 (e.g. G74D or G57D). In this study, a multiplex fluorescence real-time PCR assay, based on TaqMan minor groove binder (MGB) probes, was developed to identify C. jejuni containing mutations associated with macrolide resistance. The VS1-MGB probe was designed based on the VS1 gene and was used to identify C. jejuni. The 23S rDNA-MGB probe was designed to distinguish macrolide resistance mutations in 23S rDNA, whilst 57D-MGB and 74D-MGB were designed to detect resistance mutations in ribosomal protein L4. The specificity and accuracy of the established method were 100% identical to the classic biochemical method, mapA PCR, minimum inhibitory concentration (MIC) determination and DNA sequencing. The linear detection limitation of the method was 0.03ng/reaction genomic DNA and 3CFU/reaction bacterial colonies. In conclusion, this study provides a novel method that can specifically identify C. jejuni and accurately detect the gene mutations associated with macrolide resistance.

Analysis of single nucleotide polymorphism in human angiogenin using droplet-based microfluidics

Human angiogenin through its ribonucleolytic activity and nuclear translocation promotes angiogenesis and protects motor neurons from damage under stress conditions. Several heterozygous missense and loss of function mutations in the coding region of angiogenin have been implicated in the progression of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Every year, thousands of new patients of ALS are diagnosed, making detection of angiogenin variants an urgent necessity. In this work, we report a method of SNP detection based on offline PCR amplification of the target samples followed by bulk and droplet-based microfluidic fluorescence measurements. Our assay relied on the 5′ nuclease-based reporter excision by Taq polymerase from two differently-labeled TaqMan minor groove binder (MGB) probes, one specific for the wild type and the other for K84E single nucleotide polymorphism (SNP). The reporter-specific fluorescence signals after 30 cycles of PCR from 20 ng starting template were determined, and the wild type and the K84E SNP fluorescence intensities were approximately 3.5-times and 6-times higher compared to the no template control reaction, respectively. The results on both bulk and dropletbased microfluidic fluorescence measurements are consistent with each other, and suggest that fluorescence detection using droplet-based microfluidics can be used for the detection of SNP in angiogenin.

A novel biomarker for acute kidney injury using TaqMan-based unmethylated DNA-specific polymerase chain reaction.

There has been increasing interest in the use of circulating DNA as biomarkers for various tissue injuries, cancers, and fetal conditions. DNA methylation is a well-characterized mechanism underlying the epigenetic regulation of gene expression, and many diagnostic tests based on DNA methylation patterns have been developed. We developed a novel TaqMan-based assay for the detection of acute kidney injury using a hypomethylated promoter region of Slc22a12, a urate transporter specifically expressed in proximal tubular cells. Bisulfite sequencing analysis confirmed that the CpG islands in the promoter region of mouse Slc22a12 were preferentially hypomethylated in the kidney cortex. TaqMan minor groove binder (MGB) probes reliably discriminated the DNA fragments corresponding to the unmethylated and methylated promoter regions of Slc22a12. Plasma levels of unmethylated DNA corresponding to the Slc22a12 promoter region were undetectable at baseline and were significantly elevated after acute kidney cortex necrosis. This study showed the usefulness of the TaqMan system in discriminating methylated and unmethylated DNA fragments, and the similar strategy can be applied for establishing biomarkers for various cellular injuries or pathological conditions.

A New Method for Rapid Detection of the Mutant Allele for Chediak-Higashi Syndrome in Japanese Black Cattle

Chediak-Higashi syndrome (CHS) is an autosomal recessive hereditary disorder in Japanese Black cattle, caused by a mutation of the Lyst gene. So far, the mutation has been detected by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. However, this method is disadvantaged by its low-throughput performance. Here, we report an alternative method involving real-time PCR with TaqMan minor groove binder probes, which shortens the total assay time by more than 120 min, analyzing 10 samples in a duplicated manner. Using this method, we examined 102 Japanese Black cattle and found that 8.8% of the cattle were CHS-carriers. These data indicate that our technique is useful for routine diagnostic testing for CHS in Japanese Black cattle.

BRCA2 Promoter Hypermethylation in Sporadic Breast Cancer

<i>BRCA2</i> Promoter Hypermethylation in Sporadic Breast Cancer

Novel real-time PCR method to detect Escherichia coli O157:H7 in raw milk cheese and raw ground meat.

Raw milk, raw milk cheeses, and raw ground meat have been implicated in Escherichia coli O157:H7 outbreaks. Developing methods to detect these bacteria in raw milk and meat products is a major challenge for food safety. The aim of our study was to develop a real-time PCR assay to detect E. coli O157:H7 in raw milk cheeses and raw ground meat. Well-known primers targeting a mutation at position +93 of the uidA gene in E. coli O157:H7 were chosen, and a specific TaqMan-minor groove binder probe was designed. This probe targets another mutation, at position +191 of the uidA gene in E. coli O157:H7. The first step in the study was to evaluate the specificity of this probe with 156 different O157:H7/NM strains and 48 non-O157:H7/NM strains of E. coli. The sensitivity of the method was evaluated by pre- and postinoculation of cheeses and meat enrichments with different E. coli O157:H7 strains. All the E. coli O157:H7 isolates tested were positive, and none of the other bacteria were detected. Our results indicate that this method is sensitive enough to detect 10(2) E. coli O157:H7 isolates per ml of cheese or meat enrichment broth (24 h at 41.5° C) and is more sensitive than the International Organization for Standardization reference method. We can conclude that this new real-time PCR protocol is a useful tool for rapid, specific, and sensitive detection of E. coli O157:H7 in raw milk and raw ground meat products.

Multiplex Real-Time PCR Assay for Simultaneous Detection ofOmphalotus guepiniformisandLentinula edodes

A rapid multiplex real-time PCR assay was developed to achieve highly specific, simultaneous detection of two kinds of mushrooms, Omphalotus guepiniformis and Lentinula edodes. Primers and TaqMan minor groove binder probes were designed according to the internal transcribed spacers 1-5.8S region of rDNA and evaluated by the specificity for fruiting bodies of 17 O. guepiniformis, 16 L. edodes and samples from 57 other species. DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.00025 ng of DNA. Threshold cycle (Ct) values for raw and processed fruiting bodies and for fruiting bodies (1% (w/w)) mixed with foodstuffs or artificial gastric juice contents ranged from 17.16 to 26.60 for both examined species. This new assay proved specific to the target species, highly sensitive, and applicable to processed food samples and gastric juice contents, making it useful for rapidly identifying O. guepiniformis and L. edodes.

Analysis of acetic acid bacteria by different culture-independent techniques in a controlled superficial acetification

Three molecular techniques [denaturing gradient gel electrophoresis (DGGE-PCR), restriction fragment length polymorphism of the 16S rRNA gene amplicon (RFLP-PCR 16S rRNA) and real-time PCR (RT-PCR) with SybrGreen and with specific TaqMan-Minor Groove Binder (MGB) probes] were used to identify and monitor acetic acid bacteria (AAB) species during a controlled acetification. This process was initiated by seeding a starter culture comprising a mixture of one strain each of Acetobacter pasteurianus, Gluconacetobacter europaeus and Gluconacetobacter hansenii. Analysis at the species level indicated that the population of A. pasteurianus increased quickly, subsequently acquiring a dominant position, whereas the other two species gradually disappeared. All three methods confirmed this result. When the total AAB population was estimated, the results obtained based on summing the three species by TaqMan-MGB RT-PCR, total AAB RT-PCR and the direct microscopic count method were similar. Using TaqMan-MGB RT-PCR we were able to detect species with populations 3 log units lower than that of the major species and which could not be detected by other methods.

Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study

PurposeTo prospectively validate a protocol for noninvasive fetal sex determination in maternal plasma and demonstrate its applicability to clinical practice. MethodsPeripheral blood from 404 pregnant women undergoing prenatal invasive testing was collected from 6 to 23 weeks of gestation. Real-time PCR was performed for the SRY gene and multicopy DYS14 marker sequence located within the TSPY gene by the TaqMan minor groove binder probe assay as a first-line test. Owing to a false-positive result, amplification of repetitive motifs of the DAZ gene region was also tested as a second-line test performed in the last 232 patients enrolled in our series. A diagnostic algorithm was designed using a combination of these three markers. Fetal gender determined by noninvasive prenatal diagnosis (NIPD) was compared with that diagnosed by quantitative fluorescent PCR after invasive testing or ultrasound. ResultsA single false-positive result was obtained in the first 172 pregnancies. Reporting criteria were modified in the subsequent 232 pregnancies, giving an overall sensitivity and specificity of 100% (95% CI 99.8–100%) and 99.5% (95% CI 98.1–100%), respectively. Pregnancy outcome was obtained in all cases, including 221 male-bearing and 183 female-bearing pregnancies. ConclusionNIPD for fetal sex determination in maternal plasma is highly accurate and clinically applicable if robust reporting criteria are applied.

Novel rapid genotyping assays for neuronal ceroid lipofuscinosis in Border Collie dogs and high frequency of the mutant allele in Japan

Neuronal ceroid lipofuscinosis (NCL) constitutes a group of recessively inherited lysosomal storage diseases that primarily affect neuronal cells. Such diseases share certain clinical and pathologic features in human beings and animals. Neuronal ceroid lipofuscinosis in Border Collie dogs was first detected in Australia in the 1980s, and the pathogenic mutation was shown to be a nonsense mutation (c.619C>T) in exon 4 in canine CLN5 gene. In the present study, novel rapid genotyping assays including polymerase chain reaction (PCR)-restriction fragment length polymorphism, PCR primer-induced restriction analysis, mutagenically separated PCR, and real-time PCR with TaqMan minor groove binder probes, were developed. The utility of microchip electrophoresis was also evaluated. Furthermore, a genotyping survey was carried out in a population of Border Collies in Japan using these assays to determine the current allele frequency in Japan, providing information to control and prevent this disease in the next stage. All assays developed in the current study are available to discriminate these genotypes, and microchip electrophoresis showed a timesaving advantage over agarose gel electrophoresis. Of all assays, real-time PCR was the most suitable for large-scale examination because of its high throughput. The genotyping survey demonstrated that the carrier frequency was 8.1%. This finding suggested that the mutant allele frequency of NCL in Border Collies is high enough in Japan that measures to control and prevent the disease would be warranted. The genotyping assays developed in the present study could contribute to the prevention of NCL in Border Collies.

Develonment and application of TaqMan MGB probe real-time fluorescence quantitative PCR for rapid detection of Helicobacter hepaticus

Objective To develop a TaqMan MGB probe-based,sensitive and specific real-time fluorescence quantitative PCR assay for rapid detection of Helicobacter hepaticus.Methods Primers and probes specific toflaB gene of Helicobacter hepaticus were designed.A TaqMan MGB probe-based,real-time fluorescence quantitative PCR was established.The specificity,sensitivity and stability of the assay were assassed.Then,the established TaqMan MGB probe real-time fluorescence quantitative PCR assay was applied to detect Helicobacter hepaticus in 1081 clinical specimens during 2008-2011,compared with bacterial isolation and culture method and conventional PCR assay.Results The specificity of this established TaqMan MGB probe-based real-time fluorescence quantitative PCR was high and there were no cross-reactivity with Helicobacter pylori,Campylobacter jejuni,Clostridium piliforme,Pasteurella pneumotropica,Escherichia coli,Pseudomonas aeruginosa.The detection limits was 8.3 copies.The correlation coefficient and slope value of standard curve were 0.999 and -3.227,respectively and the efficiency of TaqMan MGB-based probe realtime fluorescence quantitative PCR assay was 100％.The TaqMan MGB-based probe real-time fluorescence quantitative PCR and conventional PCR were preformed to detect Helicobacter hepaticus in 1081 clinical specimens,a total of 86 specimens were positive for Helicobacter hepaticus.However,there was only 4 specimens were positive by bacteria isolation and culture method.The results showed that TaqMan MGB -based probe real-time fluorescence quantitative PCR for Helicobacter hepaticas was more sensitive than bacteria isolation and culture method,and it could detect Helicobacter hepaticus DNA from clinical specimens directly,and detection time is only 2 hours.Conclusion The TaqMan MGB-based probe real-time fluorescence quantitative PCR assay was a reliable,specific,sensitive and useful tool for rapid detection of Helicobacter hepaticus. Key words: Helicobacter hepaticus; Minor groove binder (MGB) probe; Real-time fluorescence quantitative PCR; Detection

Detection and identification of probable endemic fungal pathogen, Cryptococcus gattii, and worldwide pathogen, Cryptococcus neoformans, by real-time PCR

A real-time PCR method for detection and identification of Cryptococcus neoformans and Cryptococcus gattii was developed and evaluated using DNA from single-colony or koala nasal smears. Two TaqMan minor groove binder probes that distinguished between these species were designed corresponding to the internal sequences of the CAP59 gene for both species. The real-time PCR assay had 100% specificity, as assessed using 13 reference strains and 300 environmental strains. Twelve smear samples from healthy koalas were analyzed by direct real-time PCR. This method successfully detected C. gattii and C. neoformans in one and three koalas, respectively.

Detection of seasonal H3N2 influenza A virus by type-specific TaqMan minor groove binder probe assay

Despite the emergence of the pandemic H1N1 influenza A virus in 2009, seasonal H3N2 viruses continue to co-circulate in the population and may even predominate in the coming influenza season. We describe a specific minor groove binder TaqMan assay for H3N2 viruses with a detection limit of 16.5 standard DNA copies.

Development and Evaluation of a Novel Single-Nucleotide-Polymorphism Real-Time PCR Assay for Rapid Detection of Fluoroquinolone-Resistant Mycoplasma bovis

Monitoring of the susceptibility of Mycoplasma bovis field isolates to antibiotics is important for the appropriate choice of treatment. However, in vitro susceptibility testing of mycoplasmas is technically demanding and time-consuming, especially for clinical isolates, and is rarely performed in mycoplasma diagnostic laboratories. Thus, the development of methods allowing rapid real-time detection of resistant strains of M. bovis in clinical samples is a high priority for successful treatment. In this study, a novel TaqMan single-nucleotide-polymorphism (SNP) real-time PCR assay, which enables the rapid identification of M. bovis strains with different susceptibilities to fluoroquinolones, was developed and evaluated. The TaqMan SNP real-time PCR assay is based on the amplification of a 97-bp fragment of the parC quinolone resistance-determining region (QRDR) and allows the specific detection of four possible genotypes: GAC or GAT (susceptible to fluoroquinolones) and AAC or AAT (resistant to fluoroquinolones). Four TaqMan minor groove binder (MGB) probes identifying 1-base mismatches were designed and applied in a dual-probe assay with two reaction tubes. The TaqMan SNP real-time PCRs developed are highly specific for M. bovis, with a detection limit of 5 fg/microl (about 5 M. bovis genomes). In addition, all four SNP real-time PCR tests have almost the same efficiency (97.7% [GAC], 94% [AAC], 99.99% [GAT], and 98% [AAT]). Taken together, the data suggest that this SNP real-time PCR assay has potential as a routine diagnostic test for the detection of decreased susceptibility of M. bovis to fluoroquinolones.

Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients

Cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients in Brazil, and results in high mortality and morbidity, despite free access to HAART (highly active antiretroviral treatment). Molecular diagnosis based on conventional PCR (cnPCR) or real-time quantitative PCR (qrtPCR) has been indispensable for definitive diagnosis. We report here the evaluation of qrtPCR with blood and cerebrospinal fluid (CSF) samples from AIDS patients in Brazil. This prospective study was conducted for 2 years, analysing DNA samples extracted from 149 AIDS patients (98 blood and 51 CSF samples) with confirmed clinical and radiological diagnosis. The laboratory diagnosis included cnPCR (with the B22/B23 primer set) and indirect immunofluorescence (IF). For qrtPCR, two primer sets were simultaneously designed based on described genes and using a 6-carboxyfluorescein dye-labelled TaqMan MGB (minor groove binder) probe. One was B1Tg, which amplified a sequence from the B1 gene. The other was the RETg, which amplified a PCR product of the 529 bp sequence. The overall cnPCR and qrtPCR results were: positive results were observed in 33.6% (50) patients. The sensitivities were 98% for cnPCR (B22/B23), and 86 and 98% for qrtPCR (B1Tg and RETg, respectively). Negative reactions were observed in 66.4% patients. The specificities were 97% for cnPCR and qrtPCR (B1Tg), and 88.8 % for RETg. These data show that RETg PCR is highly sensitive as it amplifies a repeat region with many copies; however, its specificity is lower than the other markers. However, B1Tg PCR had good specificity, but lower sensitivity. Among the patients, 20 had blood and CSF collected simultaneously. Thus, their results permitted us to analyse and compare molecular, serological and clinical diagnosis for a better understanding of the different scenarios of laboratorial and clinical diagnosis. For nine patients with confirmed cerebral toxoplasmosis diagnosis, four scenarios were observed: (i) and (ii) negative molecular diagnosis for CSF and positive for blood with variable IF titres for the sera and CSF (negative or positive); (iii) positive molecular diagnosis with CSF and negative with blood; and (iv) positive molecular diagnosis in both samples. In the latter two situations, normally the IF titres in sera and CSF are variable. Other opportunistic infections were shown in 11 patients. Despite the IF titres in sera and CSF being variable, all of them had negative molecular diagnosis for both samples. qrtPCR allows for a rapid identification of Toxoplasma gondii DNA in patient samples; in a minority of cases discrepancies occur with the cnPCR.

Abstract 853: Prognostic significance of VEGF mRNA in pN0 patients with colorectal cancer

Abstract Objectives: Recently, we discovered isolated cytokeratin 20 (CK20) positive cells in pN0 lymph nodes (LN) from patients with colorectal cancer (CRC) and molecular dissection of this micrometastasis pathway is essential for us to understand which metastasis-related mRNA genes are involved. Using tumor metastasis quantitative polymerase chain reaction (QPCR) arrays, vascular endothelial growth factor (VEGF) mRNA has the highest median fold change in CK20 positive pN0 LN when compared to CK20 negative pN0 LN from patients with CRC (AACR Infection and Cancer abstract number B47). In this study, we validate the QPCR array results by measuring VEGF mRNA concentration in an independent set of pN0 patients with CRC and examine the prognostic significance of VEGF mRNA in the same cohort of patients. Materials and Methods: Twenty-nine paraffin-embedded pN0 LN specimens with CK20 positive cells and the same number of specimens without CK20 positive cells from patients with CRC detected by immunostaining were retrospectively retrieved from the Department of Pathology, Queen Elizabeth Hospital, Hong Kong Special Administrative Region. Total RNA was extracted from each sample for the quantification of VEGF mRNA using primers and a Taqman minor groove binder probe which labeled with a carboxyfluorescein reporter dye at the 5′-end and a nonfluorescent quencher at the 3′-end (Applied Biosystems, Foster City, USA). All 58 pN0 patients with CRC were follow-up for 48 months from their respective diagnosis for recurrent or metastatic CRC and a recurrent/metastatic curve was plotted using the median copy number of VEGF mRNA as the cut-off point. Results: The median concentration of VEGF mRNA in CK20 positive pN0 LN (35710 copy numbers, range: 5189-51240 copy numbers) was significantly higher than those in CK20 negative pN0 LN (1654 copy numbers, range: 210-4450 copy numbers) (p &amp;lt; 0.0001, Mann Whitney test). Using the median VEGF mRNA copy number (35710) of 29 CK20 positive pN0 patients with CRC as the cut-off point to plot the recurrent/metastatic curves, the time to recurrence or metastasis was significantly shorter for 14 patients with VEGF mRNA concentration &amp;gt; 35710 copy numbers (median recurrence/metastasis = 29.5 months) than for 15 patients with VEGF mRNA concentration ≤ 35710 copy numbers (p &amp;lt; 0.05, log-rank test). Conclusions: VEGF mRNA concentration is significantly correlated to the time of recurrence/metastasis and it may be able to predict the risk of recurrence/metastasis in pN0 patients with CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 853.

Rapid and Reliable Genotyping Technique for GM1 Gangliosidosis in Shiba Dogs by Real-Time Polymerase Chain Reaction with TaqMan Minor Groove Binder Probes

Real-time polymerase chain reaction (PCR) with TaqMan minor groove binder (MGB) probes was examined to establish a rapid and reliable genotyping technique for GM1 gangliosidosis in Shiba dogs. This technique was applied to DNA samples extracted from the blood, umbilical cord, or postmortem liver tissue specimens, and to DNA-containing solutions prepared from blood and saliva that had been applied to Flinders Technology Associates filter papers (FTA cards). The amplification of the targeted sequence in all the samples was sufficient to determine the genotypes of GM1 gangliosidosis. Forty-seven DNA samples that had previously been obtained from blood or tissue specimens of Shiba dogs were examined using this real-time PCR technique, and the findings were consistent with the data obtained by the earlier PCR-restriction fragment length polymorphism (RFLP) assay. In addition, the use of this new technique in combination with FTA cards for sampling could markedly shorten the time required for genotyping, as well as simplify the procedure. Furthermore, in the present study, the results of a previous epidemiological screening of 96 Shiba dogs in the Czech Republic were rechecked by this real-time PCR technique using stored crude buccal cell DNA-containing solutions directly as DNA templates. The results provided clear-cut genotyping in all the samples although the earlier PCR-RFLP assay could not determine the genotype in all cases. In conclusion, this new real-time PCR technique is a simple, rapid, and reliable choice for large-scale screening to detect an abnormal allele indicating GM1 gangliosidosis in Shiba dogs.

Detection of Pacific Cod and Capelin Roes in Alaska Pollack Roe Product by Real-Time PCR Assay

A rapid and sensitive TaqMan real-time PCR assay for the detection of Pacific cod (Gadus macrocephalus) and capelin (Mallotus villosus) roes in Alaska pollack (Theragra chalcogramma) roe product was developed. The primers and the TaqMan MGB (minor groove binder) probes were designed based on the gene encoding cytochrome b for the specific detection of Alaska pollack, Pacific cod and capelin. This real-time PCR assay had the detection limit of 0.002 ng/microL mitochondrial DNA and showed no cross-reaction with 48 other species. The calculated r(2) values of the standard curves for the three species were 1.000. This assay was applied for the detection of Pacific cod and capelin roes in mixture samples: Pacific cod or capelin roes were added to Alaska pollack roes at 0.1, 1 and 10%. The threshold cycle values were obtained from both of the mixture samples at 0.1%. Practical applicability of this assay was examined with 64 samples of Alaska pollack roe products. In all cases, the species detected from the samples corresponded with species described on the food label.