Practical Considerations in Design of Internal Amplification Controls for Diagnostic PCR Assays

Hoorfar, Jeffrey; Malorny, Burkhard; Abdulmawjood, Amir; Cook, Nigel; Wagner, Martin; Fach, Patrick

doi:10.1128/jcm.42.5.1863-1868.2004

Cited by 409 publications

(358 citation statements)

References 19 publications

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“…The competitive internal controls are suggested because the target virus RNA and IC RNA are amplified in the same RT-PCR condition. The approaches of how to generate such an IC RNA have been reviewed [81]. When using the competitive IC RNA, there are three possible outcomes of RT-PCR assays: (1) no product (the assay has failed-inhibition may be present); (2) the IC RNA, but not the virus fragment is obtained (the assay has worked, and the sample is most likely negative or viral RNA is below the detection limit); (3) the virus fragment, with or without the IC RNA fragment, is obtained (the assay has worked, and the sample is positive).…”

Section: Rt-pcrmentioning

confidence: 99%

Porcine enteric caliciviruses: Genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology

Wang

Costantini

Saif

2007

Vaccine

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Porcine enteric caliciviruses include sapoviruses and noroviruses. Porcine sapoviruses infect pigs of all ages and cause diarrhea in young pigs, whereas porcine noroviruses were detected exclusively from adult pigs without clinical signs. Importantly, certain porcine norovirus strains were genetically and antigenically related to human noroviruses. This raises public health concerns that pigs may be reservoirs for emergence of epidemic human norovirus strains. This article reviews the discovery of porcine noroviruses and sapoviruses, their classification, diagnosis, epidemiology and genetic and antigenic relatedness to human caliciviruses.

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Section: Rt-pcrmentioning

confidence: 99%

Porcine enteric caliciviruses: Genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology

Wang

Costantini

Saif

2007

Vaccine

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“…Because the same amount of armored RNA was added to each sample, the initial amount of mcyE transcript can be determined. However, although the competitive strategy is currently the most accurate in quantifying nucleic acids, the competition between the target and standard for amplification components (e.g., primers, transcriptase, dNTPs, and polymerase) can, in some instances, result in the reduction of efficiency for one or both templates (Hoorfar et al 2004). To ensure that the standard does not suppress amplification of the target sequence and vice versa, we determined the lowest possible number of pseudoviral particles that can be added to an extraction sample without causing any kind of assay interference.…”

Section: Assessmentmentioning

confidence: 99%

Use of an armored RNA standard to measure microcystin synthetase E gene expression in toxic Microcystis sp. by reverse‐transcription QPCR

Rueckert

Cary

2009

Limnology & Ocean Methods

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Microcystin is a secondary metabolic peptide toxin known to cause hepatotoxicosis and carcinogenicity in vertebrates. It is produced by various bloom-forming cyanobacterial species constituting a serious threat to the quality of freshwater reservoirs worldwide. There is an intense interest to understand the biological function of microcystin and the regulatory mechanism behind its biosynthesis in order to develop strategies for freshwater management. To date, changes in toxin production in response to various environmental parameters have been studied using direct toxin analysis. Despite extensive research over the past two decades, the function of microcystin remains poorly understood. In this study, we developed an alternative method to study the regulation of microcystin production in toxic Microcystis sp. (and alternatively in Anabaena sp.) at the level of gene expression targeting microcystin synthetase E (mcyE) transcripts. We developed a durable and ribonuclease-resistant armored RNA standard to monitor for the integrity of all nucleic acid processing steps including extraction, reverse transcription, amplification, and detection. The armored RNA was designed to consist of the mcyE target matrix with a unique internal probe-binding sequence. To validate the efficacy of the method, we conducted a comprehensive survey of mcyE expression profiling in batch cultures of Microcystis sp. during exponential, linear, and stationary growth phase. Our transcriptional analysis indicates a growth-phase-dependent expression of mcyE, with the maximal level of expression observed during mid-log growth. With progressing age of the cultures, mcyE was gradually down-regulated, with expression levels having declined by more than three orders of magnitude during the stationary growth phase.

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“…The EAC approach is time-consuming and expensive as each sample is tested twice. An alternative approach is an IAC which is simultaneously amplified in the same reaction with the target nucleic acid (Hoorfar et al 2004). …”

Section: Sapoviruses Have Been Quantitatively Detected and Characterimentioning

confidence: 99%

Application of a Competitive Internal Amplification Control for the Detection of Sapoviruses in Wastewater

et al. 2012

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Practical Considerations in Design of Internal Amplification Controls for Diagnostic PCR Assays

Cited by 409 publications

References 19 publications

Porcine enteric caliciviruses: Genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology

Porcine enteric caliciviruses: Genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology

Use of an armored RNA standard to measure microcystin synthetase E gene expression in toxic Microcystis sp. by reverse‐transcription QPCR

Application of a Competitive Internal Amplification Control for the Detection of Sapoviruses in Wastewater

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