Detecting and monitoring endophytic colonization by Pseudomonas putida BP25 in black pepper (Piper nigrum L.) using quantitative real-time PCR

2017 ◽  
Vol 26 (1) ◽  
pp. 01 ◽  
Author(s):  
V N Agisha ◽  
S J Eapen ◽  
R S Bhai ◽  
A Kumar
Keyword(s):  
Pseudomonas Putida ◽  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Colonization ◽  
Phytophthora Capsici ◽  
Pcr Primers ◽  
Black Pepper ◽  
Piper Nigrum ◽  
Quantitative Real Time Pcr ◽  
Athelia Rolfsii

A quantitative real-time PCR assay was developed to quantify Pseudomonas putida BP25, an antagonistic endophyte against a broad range of pathogens in black pepper such as Phytophthora capsici, Colletotrichum gloeosporioides, Rhizoctonia solani, Gibberella moniliformis, Athelia rolfsii and a plant parasitic nematode, Radopholus similis. The real-time PCR primers were designed based on the16S rRNA sequences of P. putida strains and specificity of the primers was confirmed. The detection limit of the assay was found to be 1 pg. The assay detected and quantified the bacterial colonization in the roots at weekly intervals after inoculation. The P. putida DNA was quantified to be 0.4 ng in roots corresponding to 5.4 log10 CFU g-1 at 7th and 14th day after inoculation (DAI). A decline in endophyte population was observed during 21st and 28th DAI and the DNA concentration ranged from 3.7-4.6 pg corresponding to 3.4-3.5 log10 CFU g-1 of root. No amplification could be obtained in stem and leaf samples. The newly developed real-time PCR could be useful for detection, quantification and monitoring of endophytic P. putida BP25 in different plant tissues.  

Detection of Cryptosporidium parvum and Giardia lamblia in Water Supply by Quantitative Real-Time PCR

2012 ◽  
Vol 518-523 ◽  
pp. 3707-3711
Author(s):  
Peng Peng ◽  
Rui Bao Jia ◽  
Yu Mei Liu ◽  
Li Li
Keyword(s):  
Water Supply ◽  
Real Time ◽  
Cryptosporidium Parvum ◽  
Real Time Pcr ◽  
Giardia Lamblia ◽  
Pcr Primers ◽  
Quantitative Real Time Pcr ◽  
Rt Pcr ◽  
High Recovery ◽  
Pcr Method

Cryptosporidium parvum and Giardia lamblia are common pathogenic protozoa in water, which pose high risk to drinking water supply. In the present study, detection of C. parvum and G. lamblia was performed by quantitative real-time PCR (RT-PCR). Pairs of PCR primers were evaluated for the detection specificity to pathogenic C. parvum and G. lamblia. The recovery of the RT-PCR detection procedure was examined and high recovery rates (i.e., more than 45％ for C. parvum and more than 50％ for G. lamblia ) were achieved. The RT-PCR method was used to detect C. parvum and G. lamblia in a secondary water supply. The results indicated the potential application of the quantitative RT- PCR method in detection of C. parvum and G. lamblia in water supply.

scholarly journals Quantitative Real-Time PCR Detection of Toxic Nodularia Cyanobacteria in the Baltic Sea

2007 ◽  
Vol 73 (7) ◽  
pp. 2173-2179 ◽  
Author(s):  
Kerttu Koskenniemi ◽  
Christina Lyra ◽  
Pirjo Rajaniemi-Wacklin ◽  
Jouni Jokela ◽  
Kaarina Sivonen
Keyword(s):  
Baltic Sea ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Gene Copy ◽  
Quantitative Real Time Pcr ◽  
The Baltic Sea ◽  
Gene Copies ◽  
Qpcr Method ◽  
The Baltic

ABSTRACT A specific quantitative real-time PCR (qPCR) method was developed for the quantification of hepatotoxin nodularin-producing Nodularia, one of the main bloom-forming cyanobacteria in the Baltic Sea. Specific PCR primers were designed for subunit F of the nodularin synthetase gene (ndaF), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30 ndaF gene copies ml−1 of seawater could be detected, and thus, the method was very sensitive. The ndaF gene copy numbers and nodularin concentrations were shown to correlate in the Baltic seawater, indicating the constant production of nodularin by Nodularia. This qPCR method for the ndaF gene can be used for detailed studies of Nodularia blooms and their formation. ndaF gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic Nodularia blooms are not only horizontally but also vertically widely distributed, and thus, the Baltic fauna is extensively exposed to nodularin.

scholarly journals MultiPriDe: automated batch development of quantitative real-time PCR primers

2008 ◽  
Vol 36 (9) ◽  
pp. 3095-3100 ◽  
Author(s):  
A. C. Ziesel ◽  
M. A. Chrenek ◽  
P. W. Wong
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Quantitative Real Time Pcr

scholarly journals More DNA and RNA of HBV SP1 splice variants are detected in genotypes B and C at low viral replication

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ka-Cheung Luk ◽  
Jeffrey Gersch ◽  
Barbara J. Harris ◽  
Vera Holzmayer ◽  
Dora Mbanya ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Real Time Pcr ◽  
Splice Variant ◽  
Splice Variants ◽  
Pcr Primers ◽  
Quantitative Real Time Pcr ◽  
Viral Loads ◽  
Hbv Genotypes ◽  
Dna And Rna

AbstractHBV produces unspliced and spliced RNAs during replication. Encapsidated spliced RNA is converted into DNA generating defective virions that are detected in plasma and associated with HCC development. Herein we describe a quantitative real-time PCR detection of splice variant SP1 DNA/RNA in HBV plasma. Three PCR primers/probe sets were designed detecting the SP1 variants, unspliced core, or X gene. Plasmids carrying the three regions were constructed for the nine HBV genotypes to evaluate the three sets, which were also tested on DNA/RNA extracted from 193 HBV plasma with unknown HCC status. The assay had an LOD of 80 copies/ml and was equally efficient for detecting all nine genotypes and three targets. In testing 84 specimens for both SP1 DNA (77.4%) and RNA (82.1%), higher viral loads resulted in increased SP1 levels. Most samples yielded < 1% of SP1 DNA, while the average SP1 RNA was 3.29%. At viral load of ≤ 5 log copies/ml, the detectable SP1 DNA varied by genotype, with 70% for B, 33.3% for C, 10.5% for E, 4% for D and 0% for A, suggesting higher levels of splicing in B and C during low replication. At > 5 log, all samples regardless of genotype had detectable SP1 DNA.

scholarly journals Bisulfite-Converted DNA Quantity Evaluation: A Multiplex Quantitative Real-Time PCR System for Evaluation of Bisulfite Conversion

2021 ◽  
Vol 12 ◽  
Author(s):  
Sae Rom Hong ◽  
Kyoung-Jin Shin
Keyword(s):  
Dna Methylation ◽  
Real Time ◽  
Conversion Efficiency ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Research Process ◽  
Bisulfite Conversion ◽  
Quantitative Real Time Pcr ◽  
Recovery Rates ◽  
Downstream Analysis

Bisulfite (BS) conversion, which includes a series of chemical reactions using bisulfite, is a prerequisite to most DNA methylation analysis methods, and thus is an essential step in the associated research process. Unfortunately, BS conversion leads to the degradation or loss of DNA, which can hinder further downstream analysis. In addition, it is well known that incomplete BS conversion is crucial, as it causes an exaggeration of the DNA methylation level, which can adversely affect the results. Therefore, there have been many attempts to measure three key features of BS conversion: BS conversion efficiency, recovery, and degradation level. In this study, a multiplex quantitative real-time PCR system named BisQuE was suggested to simultaneously analyze three important aspects of the conversion step. By adopting cytosine-free PCR primers for two differently sized multicopy regions, the short amplicon and long amplicon were obtained from both the genomic and BS-converted DNA, thus enabling the obtaining of reliable and sensitive results and the calculation of the degradation level of the conversion step. Also, probes for detecting converted/unconverted templates and C-T indicators for inducing the formula were included in this assay to quantify BS-converted DNA in order to compute the conversion efficiency and recovery. Six BS conversion kits (EZ DNA Methylation-Lightning Kit, Premium Bisulfite kit, MethylEdge® Bisulfite Conversion System, EpiJET Bisulfite Conversion Kit, EpiTect Fast DNA Bisulfite Kit, and NEBNext® Enzymatic Methyl-seq Conversion Module) were tested in 20 samples using 50 ng of genomic DNA as an input with the BisQuE. The conversion efficiency, degradation levels, as well as recovery rates of the kits were investigated. A total of 99.61–99.90% conversion efficiency was perceived for five of the kits, while the NEBNext kit showed about 94%. The lowest degradation level was shown by the NEBNext kit, whereas the other kits were quite similar. The recovery rates of the kits were found to be within the range of 18–50%. A Qubit assay was also used to compare the recovery rate of BisQuE.

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