Development of Molecular Methods to Detect Macrophomina phaseolina from Strawberry Plants and Soil

Macrophomina phaseolina is a broad-host-range fungus that shows some degree of host preference on strawberry, and causes symptoms that include crown rot and root rot. Recently, this pathogen has affected strawberry production as fumigation practices have changed, leaving many growers in California and around the world in need of accurate, rapid diagnostic tools for M. phaseolina in soil and infected plants. This study uses next-generation sequencing and comparative genomics to identify a locus that is unique to isolates within a main genotype shared by a majority of isolates that infect strawberry. This locus was used to develop a quantitative single-tube nested TaqMan polymerase chain reaction assay which is able to quantify as little as 2 to 3 microsclerotia/g of soil with 100% genotype specificity. An isothermal assay using recombinase polymerase amplification was developed from the same locus and has been validated on over 200 infected strawberry plants with a diagnostic sensitivity of 93% and a diagnostic specificity of 99%. Together, this work demonstrates the value of using new approaches to identify loci for detection and provides valuable diagnostic tools that can be used to monitor soil and strawberry plant samples for M. phaseolina.

Tracking the processing of damaged DNA double-strand break ends by ligation-mediated PCR: increased persistence of 3′-phosphoglycolate termini in SCAN1 cells

To track the processing of damaged DNA double-strand break (DSB) ends in vivo, a method was devised for quantitative measurement of 3′-phosphoglycolate (PG) termini on DSBs induced by the non-protein chromophore of neocarzinostatin (NCS-C) in the human Alu repeat. Following exposure of cells to NCS-C, DNA was isolated, and labile lesions were chemically stabilized. All 3′-phosphate and 3′-hydroxyl ends were enzymatically capped with dideoxy termini, whereas 3′-PG ends were rendered ligatable, linked to an anchor, and quantified by real-time Taqman polymerase chain reaction. Using this assay and variations thereof, 3′-PG and 3′-phosphate termini on 1-base 3′ overhangs of NCS-C-induced DSBs were readily detected in DNA from the treated lymphoblastoid cells, and both were largely eliminated from cellular DNA within 1 h. However, the 3′-PG termini were processed more slowly than 3′-phosphate termini, and were more persistent in tyrosyl-DNA phosphodiesterase 1-mutant SCAN1 than in normal cells, suggesting a significant role for tyrosyl-DNA phosphodiesterase 1 in removing 3′-PG blocking groups for DSB repair. DSBs with 3′-hydroxyl termini, which are not directly induced by NCS-C, were formed rapidly in cells, and largely eliminated by further processing within 1 h, both in Alu repeats and in heterochromatic α-satellite DNA. Moreover, absence of DNA-PK in M059J cells appeared to accelerate resolution of 3′-PG ends.

A Universal Microarray Detection Method for Identification of Multiple Phytophthora spp. Using Padlock Probes

The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp. simultaneously. A generic TaqMan polymerase chain reaction assay, which detects all known Phytophthora spp., is conducted first, followed by a species-specific PLP ligation. A 96-well-based microarray platform with colorimetric readout is used to detect and identify the different Phytophthora spp. PLPs are long oligonucleotides containing target complementary sequence regions at both their 5′ and 3′ ends which can be ligated on the target into a circular molecule. The ligation is point mutation specific; therefore, closely related sequences can be differentiated. This circular molecule can then be detected on a microarray. We developed 23 PLPs to economically important Phytophthora spp. based upon internal transcribed spacer-1 sequence differences between individual Phytophthora spp. Tests on genomic DNA of many Phytophthora isolates and DNA from environmental samples showed the specificity and utility of PLPs for Phytophthora diagnostics.

MYBL2 (B-MYB) in Cervical Cancer: Putative Biomarker

Introduction:Cervical cancer is the second most common cancer affecting women worldwide. It is characterized by chromosomal aberrations and alteration in the expression levels of many cell cycle regulatory proteins.MYBL2is a member of theMYBproto-oncogene family that encodes DNA binding proteins. These proteins are involved in cell proliferation and control of cellular differentiation.Materials and Methods:Four established cervical cancer cell lines were examined and compared with normal cervix using gene expression profiling and comparative genomic hybridization, and results were correlated to identify potential novel cervical cancer biomarkers. Results were validated using TaqMan polymerase chain reaction, and the potential role of MYBL2 as a clinical biomarker was then evaluated by immunohistochemistry on 30 tissue samples.Results:MYBL2was found to be overexpressed in the cervical cancer cell lines by gene expression profiling, and this result was confirmed using TaqMan polymerase chain reaction. Analysis of comparative genomic hybridization data indicated that chromosome 20q13.1, which encodes theMYBL2gene, was amplified in the human papillomavirus (HPV) type 16-positive CaSki and SiHa cell lines but not in the HPV-18-positive HeLa or HPV-negative C33A cell lines.Discussion:Although MYBL2 staining was predominantly absent in normal cervical epithelium, strong staining (score of 2 or 3) was identified in all cases of cervical intraepithelial neoplasia, cervical glandular intraepithelial neoplasia, and invasive cancer on immunohistochemistry. In addition, strong staining of a population of diffusely scattered single cells is identified. We postulate that these may represent so-called cancer stem-like cells.