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  Newly Published PCR Papers - Updated Daily

Real-time quantitative PCR for analysis of candidate fungal biopesticides aga... Related Articles

Real-time quantitative PCR for analysis of candidate fungal biopesticides against malaria: technique validation and first applications.

J Invertebr Pathol. 2009 Feb 6;

Authors: Bell AS, Blanford S, Jenkins N, Thomas MB, Read AF

Recent research has indicated that fungal biopesticides could augment existing malaria vector control tools. Here we present a set of methodologies to monitor the in vivo kinetics of entomopathogenic fungi in Anopheles in the presence or absence of malaria parasites using quantitative real-time PCR. Three qPCR assays were successfully developed for counting fungal genomes: "specific" assays capable of distinguishing two well characterized fungal entomopathogens Beauveria bassiana isolate IMI391510 and Metarhizium anisopliae var acridum isolate IMI330189, both of which have previously been shown to be virulent to Anopheles mosquitoes, and a "generic" fungal assay for determining any fungal burden. A fourth assay to Plasmodium chabaudi enabled quantification of co-infecting malarial parasites. All qPCR assays provide sensitive, target-specific, and robust quantification over a linear range of greater than 5 orders of magnitude (7 orders of magnitude for the fungal assays). B. bassiana growth within mosquitoes exposed to 3 different conidial challenge doses was monitored using the B. bassiana-specific assay and represents the first description of entomopathogenic fungal replication within an insect host. This revealed that, irrespective of challenge dose, after several days of relatively little replication, a sudden on-set of substantial nuclear division occurs, accompanied by physical fungal growth (hyphae) within the mosquito haemocoel shortly before death. Exposure to higher densities of conidia resulted in significantly greater pick-up by mosquitoes and to elevated fungal burdens at each time point sampled. High fungal burdens, comparable to those identified in cadavers, were attained more rapidly and mortalities occurred earlier post exposure with increasing challenge dose. The lines of research made possible by the qPCR assays described here will contribute to optimization of fungal biopesticides against malaria and other vector-borne diseases.

PMID: 19567224 [PubMed - as supplied by publisher]


Direct, real-time PCR (MethyLight) assay for methylation of O6-methylguanine-... Related Articles

Direct, real-time PCR (MethyLight) assay for methylation of O6-methylguanine-DNA methyltransferase promoter in glioma.

Chin Med J (Engl). 2009 Jun 5;122(11):1342-5

Authors: Chen G, Wu X, Yao Y, Zhou LF, Mao Y

PMID: 19567148 [PubMed - in process]


Comparative study of a new quantitative real-time PCR targeting the xylulose-... Related Articles

Comparative study of a new quantitative real-time PCR targeting the xylulose-5-phosphate/fructose-6-phosphate phosphoketolase bifidobacterial gene (xfp) in faecal samples with two fluorescence in situ hybridization methods.

J Appl Microbiol. 2009 May 26;

Authors: Cleusix V, Lacroix C, Dasen G, Leo M, Le Blay G

Aims: To detect and enumerate bifidobacteria in faeces with a new quantitative multiplex real-time PCR (qPCR) method and to compare the results obtained with fluorescence in situ hybridization (FISH) methods. Methods and Results: A multiplex qPCR assay was developed, which enabled the enumeration of Bifidobacterium spp. by targeting the bifidobacterial xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene (xfp) and total bacteria using universal Eub-primers targeting 16S rRNA gene from the domain bacteria. The qPCR assay showed high sensitivity and specificity and a low detection limit of about 2.5 x 10(3) bifidobacterial cells per gram of faeces. The qPCR results were compared with FISH combined with microscopy or flow cytometry (FCM). No statistical differences among bifidobacterial counts averages measured in adult faeces with the three methods were observed. Total bacterial count averages were higher with the FISH method coupled with microscopic analyses compared to FISH with FCM, whereas total cell numbers estimated by qPCR were intermediate between the two FISH methods. Conclusions: The new qPCR assay was shown to be sensitive, rapid and accurate for enumerating bifidobacteria in faeces. Significance and Impact of the Study: This method is a valuable alternative for other molecular methods for detecting faecal bifidobacteria, especially when their counts are below the detection limit of the FISH methods.

PMID: 19566721 [PubMed - as supplied by publisher]


[UP-PCR diversity analysis of Fusarium population isolated from greenhouse me... Related Articles

[UP-PCR diversity analysis of Fusarium population isolated from greenhouse melon soils]

Ying Yong Sheng Tai Xue Bao. 2009 Apr;20(4):857-62

Authors: Zhao BX, Gao ZG, Zhuang JH, Zhang XF, Zhao H

A total of 112 Fusarium isolates were obtained from 36 soil samples collected from the greenhouse melon fields of Liaoning Province, among which, 11 species were identified by traditional morphological classification and rDNA sequence analysis. Universally Primed PCR (UP-PCR) was conducted to analyze the 25 strains of test Fusarium isolates and 3 strains of positive control Fusarium isolates. The results indicated that a total of 73 bands appeared after amplification by using 6 primers, and 66 bands (90.4%) were polymorphic. The isolates were clustered into eight groups at the similarity of 0.736 by cluster analysis, among which, 14 isolates were clustered into one group. It was concluded that UP-PCR could present the genetic relationship and difference among Fusarium strains, being able to be used as an assistant method for Fusarium classification.

PMID: 19565767 [PubMed - in process]


primers4clades: a web server that uses phylogenetic trees to design lineage-s... Related Articles

primers4clades: a web server that uses phylogenetic trees to design lineage-specific PCR primers for metagenomic and diversity studies.

Nucleic Acids Res. 2009 Jul 1;37(Web Server issue):W95-W100

Authors: Contreras-Moreira B, Sachman-Ruiz B, Figueroa-Palacios I, Vinuesa P

Primers4clades is an easy-to-use web server that implements a fully automatic PCR primer design pipeline for cross-species amplification of novel sequences from metagenomic DNA, or from uncharacterized organisms, belonging to user-specified phylogenetic clades or taxa. The server takes a set of non-aligned protein coding genes, with or without introns, aligns them and computes a neighbor-joining tree, which is displayed on screen for easy selection of species or sequence clusters to design lineage-specific PCR primers. Primers4clades implements an extended CODEHOP primer design strategy based on both DNA and protein multiple sequence alignments. It evaluates several thermodynamic properties of the oligonucleotide pairs, and computes the phylogenetic information content of the predicted amplicon sets from Shimodaira-Hasegawa-like branch support values of maximum likelihood phylogenies. A non-redundant set of primer formulations is returned, ranked according to their thermodynamic properties. An amplicon distribution map provides a convenient overview of the coverage of the target locus. Altogether these features greatly help the user in making an informed choice between alternative primer pair formulations. Primers4clades is available at two mirror sites: http://maya.ccg.unam.mx/primers4clades/and http://floresta.eead.csic.es/primers4clades/. Three demo data sets and a comprehensive documentation/tutorial page are provided for easy testing of the server's capabilities and interface.

PMID: 19465390 [PubMed - in process]