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Response surface methodology to design a selective enrichment broth for rapid detection of Salmonella spp. by SYBR Green Ι real-time PCR

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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

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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.

Techniques for the isolation of high quality RNA from cells encapsulated in chitosan hydrogels

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Tissue Eng Part C Methods. 2013 Mar 1. [Epub ahead of print]
Yu C, Young S, Russo V, Amsden BG, Flynn LE.

Source

Queen’s University, Chemical Engineering, Kingston, Ontario, Canada; claire.yu@chee.queensu.ca.

Abstract

Extracting high quality RNA from hydrogels containing polysaccharide components is challenging as traditional RNA isolation techniques designed for cells and tissues can have limited yields and purity due to physiochemical interactions between the nucleic acids and the biomaterials. In this study, a comparative analysis of several different RNA isolation methods was performed on human adipose-derived stem cells (ASCs) photo-encapsulated within methacrylated glycol chitosan (MGC) hydrogels. The results demonstrated that RNA isolation methods with cetyl trimethylammonium bromide (CTAB) buffer followed by purification with an RNeasy® mini kit resulted in low yields of RNA, except when the samples were pre-minced directly within the buffer. In addition, genomic DNA contamination during reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was observed in the hydrogels processed with the CTAB-based methods. Isolation methods using TRIzol® in combination with one of either a Qiaex® gel extraction kit, an RNeasy® mini kit, or an extended solvent purification method extracted RNA suitable for gene amplification, with no evidence of genomic contamination. The latter two methods yielded the best results in terms of yield and amplification efficiency. Pre-digestion of the scaffolds with lysozyme was investigated as a possible means of enhancing RNA extraction from the polysaccharide gels, with no improvements observed in terms of the purity, yield, or amplification efficiency. Overall, this work highlights the application of a TRIzol® + extended solvent purification method for optimizing RNA extraction that can be applied to obtain reliable and accurate gene expression data in studies investigating cells seeded in chitosan-based scaffolds.

Evaluation of Four DNA Extraction Protocols for Brucella abortus Detection by PCR in Tissues from Experimentally Infected Cows with the 2308 Strain.

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Vector Borne Zoonotic Dis. 2013 Feb 19. [Epub ahead of print]
Vejarano MP, Matrone M, Keid LB, Rocha VC, Ikuta CY, Rodriguez CA, Salgado VR, Ferreira F, Dias RA, Telles EO, Ferreira Neto JS.

Source

Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo , São Paulo, Brazil .

Abstract

Abstract This study compared 4 protocols for DNA extraction from homogenates of 6 different organs of cows infected with the Brucella abortus 2308 strain. The extraction protocols compared were as follows: GT (guanidine isothiocyanate lysis), Boom (GT lysis with the carrying suspension diatomaceous earth), PK (proteinase K lysis), and Santos (lysis by boiling and freezing with liquid nitrogen). Positive and negative gold standard reference groups were generated by classical bacteriological methods. All samples were processed with the 4 DNA extraction protocols and amplified with the B4 and B5 primers. The number of positive samples in the placental cotyledons was higher than that in the other organs. The cumulated results showed that the Santos protocol was more sensitive than the Boom (p=0.003) and GT (p=0.0506) methods and was similar to the PK method (p=0.2969). All of the DNA extraction protocols resulted in false-negative results for PCR. In conclusion, despite the disadvantages of classical bacteriological methods, the best approach for direct diagnosis of B. abortus in organs of infected cows includes the isolation associated with PCR of DNA extracted from the cotyledon by the Santos or PK methods.

Assignment of SNP allelic configuration in polyploids using competitive allele{-}specific PCR: application to citrus triploid progeny

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Ann Bot. 2013 Feb 18. [Epub ahead of print]
Cuenca J, Aleza P, Navarro L, Ollitrault P.

Source

Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113 Moncada (Valencia), Spain.

Abstract

BackgroundPolyploidy is a major component of eukaryote evolution. Estimation of allele copy numbers for molecular markers has long been considered a challenge for polyploid species, while this process is essential for most genetic research. With the increasing availability and whole-genome coverage of single nucleotide polymorphism (SNP) markers, it is essential to implement a versatile SNP genotyping method to assign allelic configuration efficiently in polyploids.ScopeThis work evaluates the usefulness of the KASPar method, based on competitive allele-specific PCR, for the assignment of SNP allelic configuration. Citrus was chosen as a model because of its economic importance, the ongoing worldwide polyploidy manipulation projects for cultivar and rootstock breeding, and the increasing availability of SNP markers.ConclusionsFifteen SNP markers were successfully designed that produced clear allele signals that were in agreement with previous genotyping results at the diploid level. The analysis of DNA mixes between two haploid lines (Clementine and pummelo) at 13 different ratios revealed a very high correlation (average = 0·9796; s.d. = 0·0094) between the allele ratio and two parameters [θ angle = tan(-1) (y/x) and y’ = y/(x + y)] derived from the two normalized allele signals (x and y) provided by KASPar. Separated cluster analysis and analysis of variance (ANOVA) from mixed DNA simulating triploid and tetraploid hybrids provided 99·71 % correct allelic configuration. Moreover, triploid populations arising from 2n gametes and interploid crosses were easily genotyped and provided useful genetic information. This work demonstrates that the KASPar SNP genotyping technique is an efficient way to assign heterozygous allelic configurations within polyploid populations. This method is accurate, simple and cost-effective. Moreover, it may be useful for quantitative studies, such as relative allele-specific expression analysis and bulk segregant analysis.

Molecular Analysis of Spinal Muscular Atrophy: A genotyping protocol based on TaqMan(®) real-time PCR

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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.

Assessment of a semi-automated protocol for multiplex analysis of sepsis-causing bacteria with spiked whole blood samples

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Microbiologyopen. 2013 Feb 18. doi: 10.1002/mbo3.69. [Epub ahead of print]
Laakso S, Mäki M.

Source

Mobidiag Ltd., Helsinki, Finland.

Abstract

Sepsis is associated with high morbidity and mortality rates worldwide. Rapid and reliable diagnostic methods are needed for efficient and evidence-based treatment of septic patients. Recently, new molecular tools have emerged to complement the conventional culture-based diagnostic methods. In this study, we used spiked whole blood samples to evaluate together two ready-to-use molecular solutions for the detection of sepsis-causing bacteria. We spiked whole blood with bacterial species relevant in sepsis and extracted bacterial DNA with the NorDiag Arrow device, using the SelectNA Blood pathogen DNA isolation kit. DNA extracts were analyzed by the polymerase chain reaction (PCR)- and microarray-based Prove-it™ Bone and Joint assay, resulting in correctly identified bacterial species with detection limits of 11-600 colony-forming unit/mL (CFU/mL). To understand the recovery losses of bacterial DNA during the sample preparation step and the capability of the PCR- and microarray-based platform to respond to the sensitivity requirements, we also determined the analytical sensitivity of the PCR and microarray platform to be 1-21 genome equivalents for the tested bacterial species. In addition, the inclusivity of the Prove-it™ Bone and Joint assay was demonstrated with methicillin-resistant Staphylococcus aureus (MRSA) clones carrying SCCmec types I, II, IV, or V and a nontypable SCCmec type. The proof-of-concept for accurate multiplex pathogen and antibacterial resistance marker detection from spiked whole blood samples was demonstrated by the selective bacterial DNA extraction method combined with the high-throughput PCR- and microarray-based platform. Further investigations are needed to study the promising potential of the concept for sensitive, semi-automated identification of sepsis-causing pathogens directly from whole blood.

Master Mix Evaluation

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In order to improve the productivity of the genotyping processing, we think it is important to find the efficient master mixes for the PCR which leads to save our labors.

The points to pick up the right master mix we are looking at are

1. Pre-mixed and Ready to Use

2. Available on the first cycle program, within 1 hour in total

Finally we chose and tested three Master Mixes which meet our demands as following.

a. Fast Cycling PCR (Qiagen)

b. Phusion High-Fidelity PCR Master Mix (Thermo Scientific)

c. Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific)

 

Evaluation

a. Fast Cycling PCR (Qiagen) : Basically it was good, but poor yields for 1kb or longer amplicons found, even followed the instruction. There is not the 2-step protocol available in the instruction. One hour with 35 cycles. The most asset in this master mix is that almost of reactions work on the same melting temperature, at 60 degree C.

b. Phusion High-Fidelity PCR Master Mix (Thermo Scientific) : The 2-step protocol did not work for us. The 3-step protocol takes over 1 hour, so that it does not meet our demand.

c. Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific) : Basically it was as good as the Qiagen’s, and better yields for longer amplicons found than Qiagen’s. On the other hand, the 2-step protocol did not work for us as well. One hour with 35 cycles. We need to optimize the melting temperature on demands and this point is not as good as the Qiagen’s.

Prevalence of clarithromycin-resistant Helicobacter pylori in patients with chronic tonsillitis by allele-specific scorpion real-time polymerase chain reaction assay

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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.

Multiplex Picoliter-Droplet Digital PCR for Quantitative Assessment of DNA Integrity in Clinical Samples

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Clin Chem. 2013 Feb 12. [Epub ahead of print]
Didelot A, Kotsopoulos SK, Lupo A, Pekin D, Li X, Atochin I, Srinivasan P, Zhong Q, Olson J, Link DR, Laurent-Puig P, Blons H, Hutchison JB, Taly V.

Source

Université Paris Sorbonne Cité, INSERM UMR-S775, Paris, France;

Abstract

BACKGROUND: Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA.METHODS: Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3 kb. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues.RESULTS: One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants.CONCLUSIONS: The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples.