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PubMed2002

Persistent DNA contamination in competitive RT-PCR using cRNA internal standards: identity, quantity, and control.

Accurate quantification of mRNA by competitive RT-PCR demands that the quality of the cRNA internal standard be strictly controlled and that at least two criteria should be satisfied. First, genomic DNA should be removed from the total RNA being analyzed; second, template DNA should be removed from the cRNA internal standard following in vitro transcription. We observed that the routine use of RNase-free DNase I is insufficient for removing template DNA from cRNA samples and can degrade cRNA. Furthermore, reducing the template DNA before digestion, selectively extracting template DNA, and gel fractionation are all ineffective at completely eliminating template DNA contamination in cRNA standards. A strategy was developed ("inverted" competitive RT-PCR) to quantify template DNA contamination in cRNA standards. Regardless of treatment method, a small percentage of DNA contamination remained in the products of in vitro transcription. Without correction, the number of mRNA copies calculated from competitive RT-PCR is systematically overestimated. The number of template DNAs contaminating the cRNA samples was remarkably large, though as a percentage of the total cRNA, DNA contamination was small and could be easily corrected.

DNA↗
PubMed2003

[Diagnosis of dermatophytoses: conventional methods and recent molecular biological methods].

Dermatophytoses such as tinea pedis and tinea unguium are very common diseases in the field of dermatology. The diagnosis of dermatophytoses is usually performed by direct microscopy and culture. The identification of species is based on morphological features of giant culture and slide culture. However, in some cases, it is difficult to identify the species clearly because the culture shows an atypical appearance or is false negative. Therefore, several molecular biological methods have been developed for precise identification of a species. The analysis of patterns of random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) of mitochondrial DNA is useful for identifying isolates which are not clearly identifiable by conventional biological techniques. The phylogenetic analysis of dermatophytes was made by using DNA direct sequencing of nuclear ribosomal internal transcribed spacer 1 (ITS1). Sequence analysis of chitin synthase 1 (CHS 1) is a rapid tool for species level identification. We attempted the identification and viability assessment of dermatophytes based on the quantitative measurement of dermatophyte actin (ACT) mRNA. An internal fragment of the ACT, 725 to 762 bp, was isolated by PCR from the genomic DNA of dermatophytes and sequenced. ACT intron based primers were dermatophyte species-specific and primer pairs crossing the intron were dermatophyte genus-specific. The results indicated that quantification of dermatophyte ACT mRNA correlated with the results of culture and KOH examination. It is important that the identification of dermatophyte be done by combining conventional methods with molecular biological methods. In some cases results of the two methods do not correspond, and is those the fungal species needs to be re-examined.

Arthrodermataceae↗
PubMed1994

Mycobacterium smegmatis strain for detection of Mycobacterium tuberculosis by PCR used as internal control for inhibition of amplification and for quantification of bacteria.

For the detection of Mycobacterium tuberculosis by PCR, the IS6110 sequence was used. A modified target was constructed by insertion of 56 nucleotides in the IS6110 insertion element of Mycobacterium bovis BCG. This modified insertion sequence was integrated into the genome of Mycobacterium smegmatis, a mycobacterium species which does not contain the IS6110 element. When DNA from the modified M. smegmatis 1008 strain was amplified with IS6110-specific primers INS1 and INS2, a band of 301 bp was seen on agarose gel, whereas the PCR product of M. tuberculosis complex DNA was a 245-bp fragment with these primers. The addition of a small number of M. smegmatis 1008 cells to clinical samples before DNA purification enables the detection of problems which may be due to the loss of DNA in the isolation procedure or to the presence of inhibitors. The presence of inhibitors of the amplification reaction can be confirmed by the addition of M. smegmatis 1008 DNA after the DNA isolation procedure. Furthermore, competition between the different target DNAs of M. smegmatis 1008 DNA and M. tuberculosis complex DNA enables the estimation of the number of IS6110 elements in the clinical sample.

Base Sequence↗
PubMed1997

Use of the polymerase chain reaction for identification and quantification of Theileria parva protozoa in Rhipicephalus appendiculatus ticks.

The polymerase chain reaction (PCR) was adapted for detection of Theileria parva sporoblasts in Rhipicephalus appendiculatus ticks by comparison with staining of histological preparations of ticks with methyl green and pyronin (MGP). Two 32mer primers (IL174 and IL179) were used to amplify Theileria parva (Muguga isolate) DNA from the TPR 1 region of the genome by the PCR. Detection of T. parva was carried out with dissected salivary glands and whole ticks preserved in ethanol. Adult ticks which fed as nymphs on a T. parva infected calf were used in three experiments. Firstly, 70 whole ticks divided into 7 batches representing the rising and falling parasitaemia of the calf were used to show that detection of infection by the PCR was significantly correlated with MGP staining. Secondly, 120 dissected ticks were used from 4 different batches representative of the overall infection profile within the ticks to show a high correlation between PCR quantification within tick salivary glands and MGP count data of the paired gland. Thirdly, 120 ticks were used in batches selected for high and low infections. Bloodmeal contaminants from partially fed adult ticks, present in 60 out of the 120 ticks used, did not inhibit the PCR amplification of T. parva DNA. This experiment also showed a great increase in infection detection in partially fed batches of ticks compared to the untreated batches.

Animals↗
PubMed2000

Real-time polymerase chain reaction for quantification of HTLV-1 proviral load: application for analyzing aberrant integration of the proviral DNA in adult T-cell leukemia.

We describe the establishment of a real-time polymerase chain reaction (PCR) assay for the quantitative estimation of human T-cell leukemia virus type 1 (HTLV-1) proviral load using a LightCycler Technology (Roche Diagnostics, Mannheim, Germany) instrument. Proviral DNA level represents a measure of the integrated viral genome in host cells, so we applied this technique to evaluate the tumor burden in adult T-cell leukemia (ATL) patients with aberrant integration patterns of HTLV-1 detected by standard Southern blot hybridization (SBH) analysis. In 14 of our 15 ATL cases with 2 or more bands detected by SBH analysis, the ATL cells were shown to harbor multiple copies of the provirus within 1 ATL cell. This result suggests the usefulness of real-time PCR quantification for the study of the relationship between ATL pathology and HTLV-1-induced pathogenesis.

DNA, Viral↗
PubMed2002

[Structural and functional analysis of 2.2 kb spliced variant of hepatitis B virus genomes isolated from liver tissues from hepatocellular carcinoma patients].

BACKGROUND: To study the structure and function of 2.2 kb spliced variant of HBV genome from liver tissues of hepatocellular carcinoma patients. METHODS: HBV genomes were amplified by using PCR from paired hepatocellular carcinoma tissues and peritumor tissues. The 3.2 kb full-length HBV genome and 2.2 kb spliced variant were separately cloned and sequenced. Hep G2 cells were co-transfected with full-length HBV DNA and 2.2 kb spliced variant, and after transfection, HBV DNAs from intracellular core particles were harvested and specific primers were used in PCR to evaluate the interactions between spliced variant and full-length counterpart in replication. RESULTS: Semi-quantification by scanning density showed that 2.2 kb spliced variant was present in all tumor and peri-tumor samples studied. Sequence analyes revealed that the 5 terminus packaging signal for pregenomic and X and PreC/C genes were retained. When full-length HBV DNA was co-transfected with 2.2 kb, the replication signal of 3.2 kb HBV genome was increased 3-7 times. CONCLUSIONS: The 2.2 kb HBV spliced variant was present in liver tissues, and relative content was higher in tumor tissues than that in the peri-tumor tissues. This spliced variant could enhance the replication of full-length HBV genome, which suggested the possible role of the variant in the pathogenesis of development of hepatocellular carcinoma.

Adult↗
PubMed2006

Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA.

AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. RESULTS: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 multiply 10(4)/mL, 6.3 multiply 10(2)/mL and 1.6 multiply 10(3)/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 multiply 10(9)/mL, 2.08 multiply 10(6)/mL and 4.40 multiply 10(7)/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 10(5)/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. CONCLUSION: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.

Base Sequence↗
PubMed2004

Real-time quantitative RT-PCR for human telomere elongation reverse transcriptase in chronic myeloid leukemia.

Telomeres cap chromosome ends and are pivotal for DNA stability. Deregulation of the telomere stabilising enzyme telomerase in malignancy has implications in diagnosis, prognosis and therapeutics of cancer. Quantification of the expression of the telomerase catalytic subunit, hTERT, using the LightCycler TeloTAGGG hTERT Quantification kit is not optimal for analysis of chronic myeloid leukemia (CML) samples. The internal control, porphobilinogen deaminase (PBGD) is amplified in a separate tube to hTERT and has an unstable genomic localisation of 11q23. Our laboratory thus developed a real-time reverse transcriptase polymerase chain reaction which co-amplifies hTERT and either mitochondrial single-stranded DNA binding protein 1 (ssBP1) or ubiquitin C (UBC).

DNA-Binding Proteins↗
PubMed2026

Site-Specific Measurement of Meiotic Crossing-Over Rate with Droplet Digital PCR.

Understanding the frequency and distribution of meiotic crossovers (COs) is critical for both fundamental studies on meiosis and for practical applications in plant breeding, where controlling recombination can accelerate crop improvement. Determining CO rates at specific genomic loci has traditionally relied on labor-intensive methods that require the production and genotyping of large progenies. Here, we present a high-throughput protocol for site-specific quantification of meiotic COs in maize using droplet digital PCR (ddPCR). The method is based on genotyping individual pollen nuclei from hybrid plants to detect recombinant and nonrecombinant alleles at defined chromosomal intervals. By distributing several thousands of pollen nuclei into nanoliter-sized droplets and performing PCR with allele-specific fluorescent probes, this method allows precise quantification of CO frequency with high sensitivity. The protocol provides detailed guidance for nuclei isolation, probe master mix preparation, droplet generation, and data interpretation. This method can be easily adapted for use in other plants.

Journal Article↗
PubMed2006

Rapid quantitative detection of Lactobacillus sakei in meat and fermented sausages by real-time PCR.

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.

Animals↗
PubMed2006

The transcriptional regulator RovS controls the attachment of Streptococcus agalactiae to human epithelial cells and the expression of virulence genes.

Streptococcus agalactiae is part of the normal flora of the human gastrointestinal tract and also the leading cause of bacterial infections in human newborns and immunocompromised adults. The colonization and infection of different regions within the human host require a regulatory network in S. agalactiae that senses environmental stimuli and controls the formation of specific virulence factors. In the present study, we characterized an Rgg-like transcriptional regulator, designated RovS (regulator of virulence in Streptococcus agalactiae). Deletion of the rovS gene in the genome of S. agalactiae resulted in strain 6313 DeltarovS, which exhibited an increased attachment to immobilized fibrinogen and a significant increase in adherence to the eukaryotic lung epithelial cell line A549. Quantification of expression levels of known and putative S. agalactiae virulence genes by real-time PCR revealed that RovS influences the expression of fbsA, gbs0230, sodA, rogB, and the cyl operon. The altered gene expression in mutant 6313 DeltarovS was restored by plasmid-mediated expression of rovS, confirming the RovS deficiency as the cause for the observed changes in virulence gene expression in S. agalactiae. DNA electrophoretic mobility shift assays showed that RovS specifically binds to the promoter regions of fbsA, gbs0230, sodA, and the cyl operon, indicating that RovS directly regulates their expression. Deletion and mutation studies in the promoter region of fbsA, encoding the main fibrinogen receptor in S. agalactiae, identified a RovS DNA motif. Similar motifs were also found in the promoter regions of gbs0230, sodA, and the cyl operon, and alignments allowed us to propose a consensus sequence for the DNA-binding site of RovS.

Amino Acid Sequence↗
PubMed2001

Quantitation of hepatitis C virus RNA in saliva and serum of patients coinfected with HCV and human immunodeficiency virus.

The presence and the quantity of hepatitis C virus (HCV) RNA were investigated in saliva and serum of patients infected with both HCV and human immunodeficiency virus (HIV). Paired serum and saliva samples were collected from 59 HIV-HCV coinfected patients. HCV RNA was detected by nested-PCR, using primers derived from the 5' non-coding region of HCV, and positive results were quantified using the b-DNA method. HCV RNA was detected in the saliva of 22/59 (37.3%) patients, with a mean level of 1.15 x 10(6) genome equivalents/ml; there was no correlation of salivary positivity with immune status (CD4 cell count), age or HIV risk group, but there was with gender (19/38 [50%] positive results in male, compared to 3/21 [14.3%] in female, P = 0.007). HCV RNA was detected in the serum of 45/59 (76.3%) patients at a higher level (mean of 2.52 x 10(7) genome equivalents/ml) compared to saliva. Positivity was not correlated with age, gender or CD4 + cell count. There was a correlation between qualitative saliva and serum results (P = 0.003), but not between quantifications (P = 0.57). This first study reporting significant amounts of HCV RNA in saliva could have important implications for HCV epidemiology.

Adult↗
PubMedDate not supplied

Quantification of PCR products using microparticles and flow cytometry.

The analysis of genetic biomarkers has become an important tool in clinical diagnostics. This includes the identification of disease-related genetic alterations, the detection of pathogenic infective germs on DNA or RNA level and the quantification of the expression of marker genes indicating an altered physiological status. It has been previously described that the combination of polymerase chain reaction (PCR), microparticles and flow cytometry represents a universal platform technology for the routine analysis of such biomarkers. Here we demonstrate the applicability and flexibility of this technology by means of various applications. The quantification of interferon gamma (IFNG) mRNA in irradiated white blood cells is shown as well as the detection of latent infections with cytomegalovirus (CMV). Besides the quantification of single amplification products, the flow cytometric assay is also capable of analysing products of a multiplex PCR. As an example, we describe the identification of spontaneous deletions in the genome of a hybrid cell line using a co-amplified gene (RAB1) essential for the cell survival as an internal control. Furthermore, we show that the use of a green laser (532 nm, 50 mW) substantially increased the sensitivity of the assay compared to conventional flow cytometers using a 488 nm (25 mW) laser. We conclude that the analysis of PCR products using microparticles and flow cytometry fulfils the criteria of clinical routine diagnostics regarding (i) sensitivity, (ii) specificity, (iii) reproducibility and (iv) automatibility.

Animals↗
PubMed1999

Rapid and sensitive quantification of Borrelia burgdorferi-infected mouse tissues by continuous fluorescent monitoring of PCR.

The quantity of Borrelia burgdorferi organisms in tissue samples is an important determinant for infection studies in the mouse model of Lyme disease. This report presents the development of a rapid and sensitive external-standard-based PCR assay for the absolute quantification of B. burgdorferi in mouse tissue samples. The assay uses a double-stranded DNA dye to continuously monitor product formation and in less than an hour was able to quantify samples ranging up to 6 log units in concentration. The PCR efficiencies of the sample and the standard were matched by using a standard composed of purified B. burgdorferi chromosome mixed with tissue-matched mouse genome lacking bacterial DNA. Normalization of B. burgdorferi quantities to the mouse nidogen gene allowed comparison of B. burgdorferi numbers in samples isolated from different tissues and strains. PCR analysis of the chromosomal gene recA in cultured B. burgdorferi was consistent with a single recA per bacterium. The parameters defined in this assay should be applicable to quantification of other organisms, even infectious agents for which no ready source of DNA standard is available. In summary, this report presents a rapid external-standard-based PCR method for the quantification of B. burgdorferi in mouse DNA samples.

Animals↗
PubMed2005

Type I Glanzmann thrombasthenia caused by an apparently silent beta3 mutation that results in aberrant splicing and reduced beta3 mRNA.

We report a novel genetic defect in a patient with type I Glanzmann thrombasthenia. Flow cytometry analysis revealed undetectable levels of platelet glycoproteins alphaIIb and beta3, although residual amounts of both proteins were detectable in immunoblotting analysis. Sequence analysis of reversely transcribed platelet beta3 mRNA showed a 100-base pair deletion in the 3'-boundary of exon 11, that results in a frame shift and appearance of a premature STOP codon. Analysis of the corresponding genomic DNA fragment revealed the presence of a homozygous C1815T transition in exon 11. The mutation does not change the amino acid residue but it creates an ectopic consensus splice donor site that is used preferentially, causing splicing out of part of exon 11. The parents of the proband, heterozygous for this mutation, were asymptomatic and had reduced platelet content of alphaIIbbeta3. PCR-based relative quantification of beta3 mRNA failed to detect the mutant transcript in the parents and showed a marked reduction in the patient. The results suggest that the thrombasthenic phenotype is, mainly, the result of the reduced availability of beta3-mRNA, most probably due to activation of the nonsense-mediated mRNA decay mechanism. They also show the convenience of analyzing both genomic DNA and mRNA, in order to ascertain the functional consequences of single nucleotide substitutions.

Alleles↗
PubMed2004

Development of real-time PCR for detection of Mycoplasma hominis.

BACKGROUND: Mycoplasma hominis is associated with pelvic inflammatory disease, bacterial vaginosis, post partum fever, sepsis and infections of the central nervous system often leading to serious conditions. Association with development of female infertility has also been suggested, but different publications present different results. We developed a sensitive and fast diagnostic real-time PCR to test clinical samples from women undergoing laparoscopic examination before fertility treatment. To develop a test for the detection and quantification of M. hominis we selected a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gap), as a target. RESULTS: Real-time PCR was optimized to detect 10 copies of M. hominis PG21 genomic DNA. A fluorescence signal was measured for all 20 other M. hominis isolates, and melting curves analysis showed variations in the melting temperature in agreement with sequence variation in the region of the probes. There was no amplification of other mycoplasmal DNA and human DNA. Eighty-three patient cervical swab samples from infertile women were cultured for M. hominis in the BEa medium. Two of the samples (2.4%) were positive after 48 hours of incubation. The real-time PCR detected the same two samples positive, and the DNA concentrations in the clinical specimens were calculated to 37.000 copies/ml and 88.500 copies/ml, respectively. CONCLUSION: The results demonstrate that real-time PCR may prove to be a rapid alternative to the traditional cultivation method. Information on bacterial load in genital swabs can be obtained. The assay allowed detection of M. hominis in a closed system reducing the risk of contamination by amplicon carry-over.

Cervix Uteri↗
PubMed1997

PCR assay based on a microsatellite-containing locus for detection and quantification of Epichloë endophytes in grass tissue.

A PCR assay which allows detection and quantification of Epichloë endophytes in tissues of the grass Bromus erectus is described. PCR with specific primers flanking a microsatellite-containing locus (MS primers) amplified fragments 300 to 400 bp in length from as little as 1.0 pg of fungal genomic DNA in 100 ng of DNA from infected plant material. When annealing temperatures were optimized, all Epichloë and Acremonium strains tested, representing many of the known taxonomic groups, yielded an amplification product, indicating that the MS primers may be useful for in planta detection of a variety of related species, including agronomically important Acremonium coenophialum and Acremonium lolii. No fragments were generated from DNA isolates from uninfected plant material or from unrelated fungi isolated from B. erectus. For diagnostic applications, a B. erectus-specific primer pair was designed for use in multiplex PCR to allow simultaneous amplification of plant and fungal DNA sequences, providing an internal control for PCR failure caused by inhibitory plant compounds present in DNA extracts. For quantitative applications, a heterologous control template in primer binding sites complementary to the MS primers was constructed for use in competitive PCR, allowing direct quantification of Epichloë in plant DNA extracts. The fungal DNA present in infected leaves of B. erectus between 1 and 20 pg per 100 ng of leaf DNA, but the amounts of fungal DNA present in the sheath and blade of a given leaf were correlated, indicating that the degree of infection varied between plant individuals but that leaves were colonized in a uniform way.

Acremonium↗