Two methods for construction of internal amplification controls for the detection of Escherichia coli O157 by polymerase chain reaction

Abdulmawjood, Amir; Roth, Stefanie; Bülte, Michael

doi:10.1006/mcpr.2002.0431

Cited by 71 publications

(63 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Noncompetitive IPC, however, uses a completely separate target that does not directly compete with the target amplicon and has a different set of primers and probe not shared with the target amplicon for its amplification. The IPC for RRT-PCR can be introduced in a variety of ways, including transcribed RNA, armored RNA, inactivated virus, or plasmid DNA (1,6,9,10,12,14,19,27,28,31). In all cases, the IPC can be monitored in the same reaction by resolving the products on an agarose gel or using different fluorescent probes in a multiplex format with RRT-PCR.…”

mentioning

confidence: 99%

Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse Transcription-PCR with Lyophilized Reagents

et al. 2006

View full text Add to dashboard Cite

mentioning

confidence: 99%

Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse Transcription-PCR with Lyophilized Reagents

et al. 2006

View full text Add to dashboard Cite

“…The two amplicons were then differentiated by size or by the use of heterologous probes (1,4,18,23,26). A method for the production of competitive RNA controls that proved useful for the identification of RT-PCR inhibitors has been described (13).…”

mentioning

confidence: 99%

Stable and Noncompetitive RNA Internal Control for Routine Clinical Diagnostic Reverse Transcription-PCR

2004

View full text Add to dashboard Cite

Clinical diagnostic tests based on nucleic acid amplification assist with the prompt diagnosis of microbial infections because of their speeds and extremely low limits of detection. However, the design of appropriate internal controls for such assays has proven difficult. We describe a reaction-specific RNA internal control for diagnostic reverse transcription (RT)-PCR which allows extraction, RT, amplification, and detection to be monitored. The control consists of a G؉C-rich (60%) RNA molecule with an extensive secondary structure, based on a modified hepatitis delta virus genome. The rod-like structure of this RNA, with 70% intramolecular base pairing, provides a difficult template for RT-PCR. This ensures that the more favorable target virus amplicon is generated in preference to the control, with the control being detected only if the target virus is absent. The unusual structure of hepatitis delta virus RNA has previously been shown to enhance its stability and resistance to nucleases, an advantage for routine use as an internal control. The control was implemented in three nested multiplex RT-PCRs to detect nine clinically important respiratory viruses: (i) influenza A and B viruses, (ii) respiratory syncytial viruses A and B and human metapneumovirus, and (iii) parainfluenza virus types 1 to 4. The detection limits of these assays were not detectably compromised by the presence of the RNA control. During routine testing of 324 consecutive unselected respiratory samples, the presence of the internal control ensured that genuine and false-negative results were distinguishable, thus increasing the diagnostic confidence in the assay.

show abstract

“…Thus, the most critical parameter to consider is the optimal initial number of IAC copies in the diagnostic assay as it directly affects the target detection limit (Abdulmawjood et al, 2002). If used at high concentration, the IAC might not allow detection of weak inhibition which could cause false-negative results if the target is present in low concentrations (Rosenstraus et al, 1998).…”

Section: Internal Amplification Controls (Iac)mentioning

confidence: 99%

“…For a size-dependent discrimination by gel electrophoresis of target and IAC nucleic acids, chimerical IACs may be produced by deleting, or inserting sequences between the recognition primer sites (Abdulmawjood et al, 2002;Cubero et al, 2002;Müller et al, 1998;Sachadyn and Kur, 1998). A smaller size IAC can be constructed by deleting an internal sequence fragment.…”

Section: Construction Of An Iacmentioning

confidence: 99%

Molecular methodology in Food Microbiology diagnostics: trends and current challenges

Rodrı́guez-Lázaro

Hernández

2006

13th World Congress of Food Science &Amp; Technology

View full text Add to dashboard Cite

Foodborne diseases are among the most serious public health concerns worldwide. Consequently, the application of microbiological controls within the quality assessment programs in the food industry is a premise to minimize the risk of infection for the consumer. Classical microbiological methods involve, in general, the use of appropriate pre-enrichment and enrichment, isolation on selective media, and subsequent confirmation using morphological, biochemical and/or serological tests. They are laborious, time consuming and not always reliable. A number of alternative, rapid and sensitive molecular methods for the detection, identification and quantification of foodborne pathogens have been developed to overcome these drawbacks. However, until now, they have shown low practical implementation for monitoring and microbiological control. Amplification techniques have a number of advantages including improved speed, excellent detection limit, specificity, sensitivity and potential for quantification as well as easy automation. Major drawbacks are the cost of the analysis and the qualified personnel needed to carry out the experiments, plus the lack of standardisation of protocols approved by normalisation bodies. Moreover, the cost of reagents is decreasing and more qualified people are available to perform this kind of analysis. Efforts on standardisation and normalisation of these techniques are a key priority.The molecular methods, in conclusion, are a promising alternative that can substitute or complement the current reference methods in food microbiology diagnostics. More suitable and reliable results can be achieved in terms of speed and precision, and can detect and enumerate specifically viable microorganisms.

show abstract

Two methods for construction of internal amplification controls for the detection of Escherichia coli O157 by polymerase chain reaction

Cited by 71 publications

References 7 publications

Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse Transcription-PCR with Lyophilized Reagents

Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse Transcription-PCR with Lyophilized Reagents

Stable and Noncompetitive RNA Internal Control for Routine Clinical Diagnostic Reverse Transcription-PCR

Molecular methodology in Food Microbiology diagnostics: trends and current challenges

Contact Info

Product

Resources

About