scholarly journals 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

2010 ◽  
Vol 108 (1) ◽  
pp. 181-193 ◽  
Author(s):  
V. Cleusix ◽  
C. Lacroix ◽  
G. Dasen ◽  
M. Leo ◽  
G. Le Blay
Keyword(s):  
In Situ Hybridization ◽  
Comparative Study ◽  
Fluorescence In Situ Hybridization ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Faecal Samples ◽  
Pcr Targeting

scholarly journals Improved Enumeration of Lactic Acid Bacteria in Mesophilic Dairy Starter Cultures by Using Multiplex Quantitative Real-Time PCR and Flow Cytometry-Fluorescence In Situ Hybridization

2006 ◽  
Vol 72 (6) ◽  
pp. 4163-4171 ◽  
Author(s):  
Udo Friedrich ◽  
Jan Lenke
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Real Time ◽  
Real Time Pcr ◽  
Starter Cultures ◽  
Calcium Citrate ◽  
Quantitative Real Time Pcr ◽  
Dairy Starter Cultures

ABSTRACT Nucleic acid-based assays were developed to enumerate members of the three taxa Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, and Leuconostoc spp. in mesophilic starter cultures. To our knowledge the present is the first study to present a multiplex quantitative PCR (qPCR) strategy for the relative enumeration of bacteria. The multiplex qPCR strategy was designed to quantify the target DNA simultaneously relative to total bacterial DNA. The assay has a high discriminatory power and resolves concentration changes as low as 1.3-fold. The methodology was compared with flow cytometric fluorescence in situ hybridization (FLOW-FISH) and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal)-calcium citrate agar-based plate counting. For enumeration by FLOW-FISH, three new probes having the same specificity as the qPCR assay were designed and established. A combination with flow cytometry greatly reduced the time consumed compared to manual enumeration. Both qPCR and FLOW-FISH yielded similar community compositions for 10 complex starter cultures, with all detected subpopulations being highly significantly correlated (P < 0.001). Correlations between X-Gal-calcium citrate agar-based CFU and qPCR-derived counts were highly significant (P < 0.01 and P < 0.001, respectively) for the number of acidifiers versus L. lactis subsp. cremoris and for Leuconostoc spp. as quantified by the two techniques, respectively. This confirmed that most acidifiers in the studied PROBAT cultures are members of L. lactis subsp. cremoris. Quantitative real-time PCR and FLOW-FISH were found to be effective and accurate tools for the bacterial community analysis of complex starter cultures.

Quantification of an Eikelboom type 021N bulking event with fluorescence in situ hybridization and real-time PCR

2005 ◽  
Vol 68 (5) ◽  
pp. 695-704 ◽  
Author(s):  
Han Vervaeren ◽  
Kurt De Wilde ◽  
Jorg Matthys ◽  
Nico Boon ◽  
Lutgarde Raskin ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Real Time ◽  
Real Time Pcr

Low-level copy gain versus amplification of myc oncogenes in medulloblastoma: utility in predicting prognosis and survival

2009 ◽  
Vol 3 (1) ◽  
pp. 61-65 ◽  
Author(s):  
Hidehiro Takei ◽  
Yummy Nguyen ◽  
Vidya Mehta ◽  
Murali Chintagumpala ◽  
Robert C. Dauser ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Real Time ◽  
Real Time Pcr ◽  
Large Cell ◽  
Histological Subtype ◽  
Quantitative Real Time Pcr ◽  
Low Level ◽  
Kaplan Meier ◽  
High Level

Object Medulloblastoma (MB) is a malignant embryonal tumor of the cerebellum. Amplification of c-myc or N-myc is infrequently identified and, when present, is often associated with the large cell/anaplastic (LC/A) phenotype. The frequency of low-level copy gain of myc oncogenes and its relationship to prognosis of MB has not been explored. Methods Archival cases of MB were histologically reviewed and classified into 3 major subtypes: classic, nodular, and LC/A. Using quantitative real-time polymerase chain reaction (PCR), the authors analyzed 58 cases with a pure histological subtype for the copy number (CN) of myc (c-myc and N-myc) oncogenes. Cases with > 5-fold CN were further analyzed using the fluorescent in situ hybridization (FISH) assay. Kaplan-Meier survival analysis was performed. Results A > 5-fold myc CN was noted in 5 (20.8%) of 24 LC/A, 1 (5.3%) of 19 classic, and 2 (13.3%) of 15 nodular subtypes. In a significant number of tumors (14 [56%] of 24 LC/A, 13 [68%] of 19 classic, and 10 [67%] of 15 nodular MBs) the CN was > 2-fold but < 5-fold. High-level amplification, defined as > 10-fold CN, was only seen in the LC/A subtype (5 cases), although moderate amplification (> 5-fold but < 10-fold) could be detected in other histological subtypes. Fluorescence in situ hybridization readily detected most cases corresponding to tumors with > 5-fold amplicon CN by quantitative real-time PCR, and could detect all 5 cases with > 10-fold CN by quantitative real-time PCR. The group of patients with > 5-fold myc amplicon CN showed significantly shorter survival than those with < 5-fold CN (p = 0.045), independent of histological subtype. Conclusions Since FISH could easily detect most cases in the moderate-to-high myc gene amplification (> 5-fold CN) group, the FISH assay has utility in detecting subsets of MB with poorer prognosis.

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