Find out what roles both qPCR and dPCR play in today’s academic, industry, and clinical research labs.
George Karlin-Neumann, Ph.D. , Rachel Scott, Ph.D.
Tag Archives: qPCR
High-resolution melting analysis for bird sexing: a successful approach to molecular sex identification using different biological samples
Mol Ecol Resour. 2013 Feb 25. doi: 10.1111/1755-0998.12081. [Epub ahead of print]
Morinha F, Travassos P, Seixas F, Santos N, Sargo R, Sousa L, Magalhães P, Cabral JA, Bastos E.
Source
Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes e Alto Douro (IBB/CGB-UTAD), Quinta de Prados, P.O. Box 1013, Vila Real, 5001-801, Portugal; Laboratory of Applied Ecology, Centre for the Research and Technology of Agro-Environment and Biological Sciences (CITAB), University of Trás-os-Montes e Alto Douro, Quinta de Prados, P.O. Box 1013, Vila Real, 5001-801, Portugal.
Abstract
High-resolution melting (HRM) analysis is a very attractive and flexible advanced post-PCR method with high sensitivity/specificity for simple, fast and cost-effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real-time PCR systems, along with improved saturating DNA-binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sex-specific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed-tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real-time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing.
Response surface methodology to design a selective enrichment broth for rapid detection of Salmonella spp. by SYBR Green Ι real-time PCR
Appl Microbiol Biotechnol. 2013 Mar 7. [Epub ahead of print]
Zhang Q, Chen T, Yang S, Wang X, Guo H.
Source
Zhejiang Province Key Lab for Food Safety, Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, Zhejiang, China, yanyan0014@sina.com.
Abstract
In order to meet dominant growth of Salmonella spp. in a composed system of five pathogens for accurate detection, designing an appropriate selective enrichment broth was clearly needed. First, we built a high-throughput assay procedure based on SYBR Green Ι real-time PCR, which possessed the necessary specificity for Salmonella spp., a good linear standard curve with typical R 2 value (0.9984) and high amplification efficiency (99.0 %). Further, for the larger target biomass in the mixed microflora, acarbose, LiCl and bile salt were selected to optimize their concentrations using response surface methodology (RSM). A central composite design was employed to collect the data and fit the response. A quadratic polynomial model was derived by computer simulation. Statistical analysis was carried out to explore the action and interaction of the variables on the response. In the end, a novel broth (Sal-5) was formulated to allow the efficient enrichment of Salmonella spp. and inhibit the growth of other tested strains. A detection platform was developed, including selective enrichment in Sal-5, DNA extraction by the boiling lysis method and real-time PCR test based on SYBR Green Ι. This work could extend the application of RSM and real-time PCR in the design of other selective enrichment media for common pathogens.
Comparison of DNA Extraction Kits for Detection of Burkholderia pseudomallei in Spiked Human Whole Blood Using Real-Time PCR
PLoS One. 2013;8(2):e58032. doi: 10.1371/journal.pone.0058032. Epub 2013 Feb 27.
Podnecky NL, Elrod MG, Newton BR, Dauphin LA, Shi J, Chawalchitiporn S, Baggett HC, Hoffmaster AR, Gee JE.
Source
Bacterial Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
Abstract
, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of DNA extracted by each kit was performed using the specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (C ) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.
Molecular Analysis of Spinal Muscular Atrophy: A genotyping protocol based on TaqMan(®) real-time PCR
Genet Mol Biol. 2012 Dec;35(4 (suppl)):955-9. Epub 2012 Dec 18.
de Souza Godinho FM, Bock H, Gheno TC, Saraiva-Pereira ML.
Source
Laboratório de Identificação Genética, Centro de Pesquisa Experimental e Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, RS, Brazil. ; Programa de Pós-Graduação em Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
Abstract
Spinal muscular atrophy (SMA) is an autosomal recessive inherited disorder caused by alterations in the survival motor neuron I (SMN1) gene. SMA patients are classified as type I-IV based on severity of symptoms and age of onset. About 95% of SMA cases are caused by the homozygous absence of SMN1 due to gene deletion or conversion into SMN2. PCR-based methods have been widely used in genetic testing for SMA. In this work, we introduce a new approach based on TaqMan(®)real-time PCR for research and diagnostic settings. DNA samples from 100 individuals with clinical signs and symptoms suggestive of SMA were analyzed. Mutant DNA samples as well as controls were confirmed by DNA sequencing. We detected 58 SMA cases (58.0%) by showing deletion of SMN1 exon 7. Considering clinical information available from 56 of them, the patient distribution was 26 (46.4%) SMA type I, 16 (28.6%) SMA type II and 14 (25.0%) SMA type III. Results generated by the new method was confirmed by PCR-RFLP and by DNA sequencing when required. In conclusion, a protocol based on real-time PCR was shown to be effective and specific for molecular analysis of SMA patients.
Prevalence of clarithromycin-resistant Helicobacter pylori in patients with chronic tonsillitis by allele-specific scorpion real-time polymerase chain reaction assay
Laryngoscope. 2013 Feb 12. doi: 10.1002/lary.23777. [Epub ahead of print]
Naserpour Farivar T, Najafipour R, Johari P.
Source
Cell and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran.
Abstract
OBJECTIVES/HYPOTHESIS:
To investigate the allelic prevalence of resistance to clarithromycin in the DNA of clinical isolates of Helicobacter pylori obtained from biopsy specimens of patients with chronic tonsillitis by Scorpion real-time polymerase chain reaction (PCR).
STUDY DESIGN:
Pathologic specimens of patients with chronic tonsillitis were used for rapid urease test, and blocks of paraffin-embedded tonsillar tissue were used for McMullen staining, rapid urease test, and Scorpion real-time PCR test.
METHODS:
A total of 103 biopsy samples were obtained from patients with chronic tonsillitis and examined for the presence of clarithromycin resistant H. pylori. Modified McMullen staining and rapid urease test were done on the all the samples. The DNA of specimens was extracted from the pathology blocks, and Scorpion real-time PCR was performed on a final volume of 25 μL.
RESULTS:
Of 103 biopsy specimens, 22 samples were identified as infected by H. pylori, of which none were sensitive to clarithromycin. One had the A2143G genotype, and four had the A2142G genotype. Two had a mixed sensitive and the A2143G genotype, and five had a mixed sensitive and A2142G genotype. One strain had a mixed genotype of sensitive, A2143G, and A2142G.
CONCLUSIONS:
The reported rate of resistance to clarithromycin is of great variation among H. pylori strains isolated from specimens in different countries. Our study showed that the most prevalent genotypes in our H. pylori-positive specimens was A2142G followed by A2143G, which is different from reported results of allele-specific genotyping of H. pylori strains isolated from gastric biopsy and may be a result of cross-resistance to erythromycin and other macrolides.
A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva
Lab Chip. 2013 Feb 1. [Epub ahead of print]
Oblath EA, Henley WH, Alarie JP, Ramsey JM.
Source
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. jmramsey@unc.edu.
Abstract
A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100-125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (8-12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device.
Fast and specific dermatophyte detection by automated DNA extraction and real-time PCR
Clin Microbiol Infect. 2013 Jan 24. doi: 10.1111/1469-0691.12153. [Epub ahead of print]
Bergman A, Heimer D, Kondori N, Enroth H.
Source
Department of Clinical Microbiology, Unilabs AB, Skövde, Sweden.
Abstract
The aim of this study was to develop and validate a rapid and sensitive real-time PCR method for detection of all known species of dermatophytes, including identification of Trichophyton rubrum and Trichophyton interdigitale. Fungal DNA was extracted directly from clinical samples by using a pre-lysis step, followed by automated DNA extraction on the MagNA Pure Compact. In total, 202 clinical samples were examined by both conventional culture and by the new PCR method. In 103 (51%) of the samples fungal nucleic acid was detected by PCR, while only 79 (39%) were found to be positive by culture. Out of 103 PCR-positive clinical samples, 94 (91%) were identified as T. rubrum and eight (8%) as T. interdigitale. This real-time PCR is far more sensitive and 2-4 weeks faster than conventional culture for detection of dermatophytes present in clinical samples.