A molecular beacon-based real time NASBA assay for detection of Listeria monocytogenes in food products: Role of target mRNA secondary structure on NASBA design

2007 ◽  
Vol 68 (3) ◽  
pp. 623-632 ◽  
Author(s):  
Anna Nadal ◽  
Anna Coll ◽  
Nigel Cook ◽  
Maria Pla
Keyword(s):  
Listeria Monocytogenes ◽  
Secondary Structure ◽  
Real Time ◽  
Molecular Beacon ◽  
Food Products ◽  
Mrna Secondary Structure ◽  
Target Mrna ◽  
Nasba Assay

scholarly journals Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

2005 ◽  
Vol 71 (11) ◽  
pp. 7113-7116 ◽  
Author(s):  
Khaled H. Abd El Galil ◽  
M. A. El Sokkary ◽  
S. M. Kheira ◽  
Andre M. Salazar ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Nucleic Acid Sequence ◽  
The Real ◽  
Nasba Assay ◽  
Environmental Pathogens

ABSTRACT A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.

scholarly journals A molecular beacon-based real-time NASBA assay for detection ofMycobacterium aviumsubsp.paratuberculosisin water and milk

2004 ◽  
Vol 237 (1) ◽  
pp. 119-126 ◽  
Author(s):  
David Rodríguez-Lázaro ◽  
Joy Lloyd ◽  
Arnold Herrewegh ◽  
John Ikonomopoulos ◽  
Martin D'Agostino ◽  
...  
Keyword(s):  
Real Time ◽  
Molecular Beacon ◽  
Nasba Assay

255 GENE EXPRESSION OF Cox 5a, 5b, AND 6b1 AND THEIR ROLES IN PRE-IMPLANTATION OF MOUSE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 235
Author(s):  
S.-E. Lee ◽  
X.-Y. Li ◽  
X.-S. Cui ◽  
N.-H. Kim
Keyword(s):  
Rna Interference ◽  
Mrna Expression ◽  
Real Time ◽  
Oocyte Maturation ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Target Mrna ◽  
Protein Levels

Despite clear evidence of regulation of mitochondrial respiration by nuclear encoded genes, cytochrome oxidase (Cox), little information is available on their expression and functional roles during early embryonic development. To examine the role of Cox in oocyte maturation and embryogenesis, we first characterized mRNA and protein levels of nuclear encoded genes, Cox 5a, 5b, and 6b1, in mouse oocytes and during early embryogenesis, using real-time RT-PCR and immunocytochemistry. We then examined the possible role of these genes in oocyte maturation and pre-implantation development using RNA interference analysis. The relative abundances of Cox 5a, 5b, and 6b1 transcripts was measured by real time RT-PCR. After normalization by comparison to histone H2a mRNA levels, the mRNA expression of Cox 5a, 5b, and 6b1 were found to be considerable in mature oocytes and zygotes, but reduced slightly in 2-cell embryos. From the 2-cell to the blastocyst stage, mRNA expression is dependent on the number of blastomeres, as expression increases only gradually with development. Immunocytochemical studies revealed that Cox 5a, 5b, and 6b1 proteins were expressed in all blastomeres of the blastocyst. Injection of Cox 5a, 5b, or 6b1 siRNA into GV stage oocytes decreased expression of the target mRNA specifically, while not affecting the expression of mRNAs for the other subunits in mature oocytes. Similarly, each siRNA injection into zygotes specifically reduced target mRNA expression at the 2-cell, morula and blastocyst stages (P < 0.05). Silencing of mRNA expression by RNA interference (siRNA) did not inhibit oocyte maturation or developmental events up to the morula and blastocyst stages. The expression level of mtDNA9, as well as overall levels of mitochondrial mRNAs, was not different following injection of siRNA for Cox 5a, 5b, or 6b1. However, it is evident that the number of mitochondria in siRNA treated blastocysts was greatly reduced, and they appeared to be morphologically abnormal. Significantly higher apoptosis and lower cell numbers were observed in siRNA treated blastocysts. Real time RT PCR revealed that silencing of Cox 5a, 5b, and 6b1 decreased mRNA and protein levels of E-cadherin. These results suggest that the Cox subunits, Cox 5a, 5b, and 6b1, play an important role in mitochondrial function during pre-implantation development. This work was funded by a grant from the National Research Laboratory Program in Korea.

scholarly journals mRNA structure regulates protein expression through changes in functional half-life

2019 ◽  
Vol 116 (48) ◽  
pp. 24075-24083 ◽  
Author(s):  
David M. Mauger ◽  
B. Joseph Cabral ◽  
Vladimir Presnyak ◽  
Stephen V. Su ◽  
David W. Reid ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Protein Expression ◽  
Codon Usage ◽  
Half Life ◽  
Order Structure ◽  
Messenger Rnas ◽  
Mrna Secondary Structure ◽  
Modified Nucleotides ◽  
Highly Correlated

Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used 2 approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs, to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly expressed mRNAs contained a highly structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide variety of primary sequences. Using a set of eGFP mRNAs with independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e., mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability.

Real-time molecular beacon NASBA for rapid and sensitive detection of norovirus GII in clinical samples

2009 ◽  
Vol 55 (12) ◽  
pp. 1375-1380 ◽  
Author(s):  
Safaa Lamhoujeb ◽  
Hugues Charest ◽  
Ismail Fliss ◽  
Solange Ngazoa ◽  
Julie Jean
Keyword(s):  
Real Time ◽  
Molecular Beacon ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reading Frame ◽  
Viral Loads ◽  
Reverse Transcription Pcr ◽  
Highly Sensitive ◽  
Nasba Assay ◽  
Norovirus Gii

To improve the sensitivity and efficiency of the real-time nucleic acid sequence based amplification (NASBA) assay targeting the open reading frame 1–2 (ORF1–ORF2) junction of the norovirus (NoV) genome, a selection of clinical samples were analyzed. The assay results were compared with those of TaqMan and conventional reverse transcription PCR (RT-PCR) and a commercial enzyme-linked immunoassay (ELISA) for the specific detection of GII NoV in 96 fecal samples. Based on end-point dilution, the two real-time assays had similar sensitivities (0.01 particle detectable units), two log10cycles greater than that of conventional RT-PCR. GII NoV was detected in 88.54% of the samples by real-time NASBA, in 86.46% by TaqMan RT-PCR, in 81.25% by conventional RT-PCR, and in 65.7% by ELISA. The two real-time assays were in agreement for 88.5% of the samples. These results demonstrate that real-time NASBA with a molecular beacon probe is highly sensitive, accurate, and specific for NoV detection in clinical samples. Applying this technique to samples with complex matrix and low viral loads, such as food and environmental samples, could be useful for the detection of NoVs and will improve the prevention of NoV outbreaks.

A duplex real-time NASBA assay targeting serotype-specific gene for rapid detection of viable S. enterica serovar Paratyphi C in retail foods of animal origin

2022 ◽  
Author(s):  
Zhai Ligong ◽  
Liu Hongxia ◽  
Li Junjie ◽  
Zhaoxin Lu ◽  
Xiaomei Bie
Keyword(s):  
Real Time ◽  
Molecular Beacon ◽  
Detection Method ◽  
Simultaneous Detection ◽  
Animal Origin ◽  
Specific Gene ◽  
Salmonella Spp ◽  
Viable Cells ◽  
Food Of Animal Origin ◽  
Nasba Assay

Salmonella enterica serovars Paratyphi C is highly adapted to humans and can cause a typhoid-like disease with high mortality rates. In this study, three serovar-specific genes were determined for S. Paratyphi C, SPC_0871，SPC_0872, and SPC_0908, by comparative genomics method. Based on SPC_0908 and xcd gene for testing Salmonella spp., we have developed a duplex real-time nucleic acid sequence-based amplification (real-time NASBA) with molecular beacon approach for simultaneous detection of viable cells of Salmonella spp. and serotype Paratyphi C. The test selectively and consistently detected 53 Salmonella spp. (representing 31 serotypes) and 18 non-Salmonella strains. Additionally, the method showed high resistance to interference by natural background flora in pork and chicken samples. The sensitivity of the established approach was determined to be 4.89 CFU/25 g in artificially contaminated pork and chicken samples after pre-enrichment. We propose this NASBA-based protocol as a potential detection method for Salmonella spp. and serotype Paratyphi C in food of animal origin.

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