Checklist for optimization and validation of real‐time PCR assays

Raymaekers, M.; Smets, Rita; Maes, Brigitte; Cartuyvels, Reinoud

doi:10.1002/jcla.20307

Cited by 159 publications

(121 citation statements)

References 68 publications

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“…Our results confirmed that enteric viruses play an important role in pediatric diarrhea, and further that the most common viral agent associated with gastroenteritis is NoV, followed by RV, HAstrV, HBoV, SaV, HPeV, AdV, and SalV. The prevalence rates of RV, SaV, HPeV, AdV, and HBoV were similar to those reported previously [32, 34-36]. However, the detection rates of 40.5 and 18.87% for NoV and HAstrV, respectively, in this study were higher than those reported previously in Italy [37-39] but similar to those in the recent study of Rovida et al [40].…”

Section: Discussionsupporting

confidence: 91%

Aichivirus in Children with Diarrhea in Northern Italy

Bergallo¹,

Galliano²,

Montanari³

et al. 2017

Intervirology

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Objective: Since its discovery, Aichivirus (AiV) A has been detected, with an incidence of 0.9–4.1%, primarily when studying outbreaks of diarrhea in children or young adults. In this paper, we report the first detection of AiV in Piedmont, Italy, in pediatric patients. Methods: A total of 159 fecal specimens (from 96 males and 63 females) previously screened for rotaviruses, adenoviruses, noroviruses, human parechoviruses, saliviruses, and sapoviruses were collected from infants and children with acute gastroenteritis. Results: The most commonly detected virus was norovirus GII (33.80%), fol lowed by rotavirus (21.30%), astrovirus (18.87%), boca virus (13.92%), sapovirus (10.90%), parechovirus (8%), norovirus GI (6.70%), adenovirus (1%), and salivirus (0.52%). Real-time polymerase chain reaction detected AiV A in 1 (0.62%) case subjects. AiV A was detected in monoinfection only in January. Conclusions: Our results indicate that AiV may be associated with a limited number of diarrhea cases in pediatric patients.

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Section: Discussionsupporting

confidence: 91%

Aichivirus in Children with Diarrhea in Northern Italy

Bergallo¹,

Galliano²,

Montanari³

et al. 2017

Intervirology

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“…The development of a "in-house" Real Time RT-PCR assay, available for diagnostic routine, requires an analogous process applied to marked and approved commercial kit for diagnostic use (4,8). This is realized by the standardization and optimization of the amplification protocol; however, also RNA extraction and reverse transcription protocols should be considered in the method assessment.…”

Section: Discussionmentioning

confidence: 99%

Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR

et al. 2010

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Real Time RT-PCR developed in recent years represents an useful tool in the diagnosis of RNA viruses. In order to accurately quantify and normalize a RNA target, efficiency of reverse-transcription must be considered. In this study a cRNA-standard based quantitative Real Time RT-PCR have been developed for HRV quantification on bronchoalveolar lavage (BAL) specimens. Results has been compared to a quantitative plasmid standard-based Real Time RT-PCR previously developed by us.Large amount of pHRV was linearized and purified. Blunt ends were generated and cRNA production was carried out. Dilutions of cRNA were generated and dynamic range, intraand inter-test variability, sensitivity and limit of detection were evaluated. Sixty-seven BAL, previously resulted positive to our plasmid standard-based method, were evaluated using cRNA-standard quantification. cRNA curve showed a broad dynamic range with a good intra-and inter-test variability, with an average of 3.23 threshold cycles more in comparison to plasmid standard-based curve. In terms of specimen quantification, a difference of 1.07log was found, showing a significant underrate using plasmid standard-based quantification.The method for cRNA-standard construction seems more suitable for quantification of RNA viruses, in order to normalize the quantification in reverse-transcription.

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“…All assays were carried out on a Stratagene Mx3005P thermal cycler. Assay characteristics, including reaction efficiency, dynamic range, intraand interassay variability, and limit of detection, were determined as described previously (7). The sensitivity of the assay for detecting genomic DNA from diverse A. baumannii strains was determined using purified genomic DNA (QIAamp DNA minikit; Qiagen) from 20 clonally distinct clinical isolates, as determined by pulsed-field gel electrophoresis or repetitive element PCR (REP-PCR).…”

mentioning

confidence: 99%

Quantitative Real-Time PCR for Detection of Acinetobacter baumannii Colonization in the Hospital Environment

et al. 2012

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A real-time PCR assay was developed for detecting the presence of Acinetobacter baumannii on hospital equipment and compared to conventional bacterial culture using 100 hospital environmental samples. The real-time PCR detected contaminated surfaces in 4 h with high sensitivity (100%) compared to conventional culture. Thirty-eight percent of samples were positive by real-time PCR and negative by bacterial culture (false positives), possibly indicating the widespread presence of bacterial DNA that is not associated with viable bacteria. N osocomial infections caused by drug-resistant bacteria represent an important clinical challenge. Acinetobacter baumannii has become one of the most problematic causative agents of nosocomial infections due to its remarkable ability to survive on hospital surfaces and acquire antibiotic resistance, resulting in the global emergence of multidrug-resistant strains with resistance to multiple antibiotic classes (5). A. baumannii has been especially problematic in critically ill patients in the intensive care setting, as it is an important cause of ventilator-associated pneumonia and bacteremia. In this context, patients are exposed to A. baumannii via contact with contaminated hospital equipment or by contact with hospital personnel carrying the bacteria. A number of studies have demonstrated widespread contamination with A. baumannii on hospital environmental surfaces, most notably in intensive care units (ICUs) (1,4,8,9).Environmental surveillance protocols have been employed for the identification of hospital equipment colonized by A. baumannii so that appropriate decontamination procedures can be carried out (1,4,8,9). Since these surveillance methods employ conventional bacterial culture to determine the presence of A. baumannii, definitive species identification can require between 24 and 48 h. Nucleic acid-based tests, such as real-time PCR, have been employed for the identification of numerous bacterial pathogens (2); however, to our knowledge this technique has not been applied to identifying contaminated hospital equipment. The objective of the present study was to develop a real-time PCR for identifying hospital surfaces colonized by A. baumannii.A real-time PCR assay was developed using TaqMan chemistry for the amplification of nucleotides 774 to 859 of the outer membrane protein A gene (ompA; accession number AY485227). The ompA gene was chosen because it is present in all sequenced genomes of A. baumannii available in the public domain (as of March 2010), and the sequences chosen for the primers and probe correspond to regions highly conserved between published A. baumannii ompA sequences (100% sequence identity). The primers OmpA Forward (5=-TCTTGGTGGTCACTTGAAGC-3=) and Ompa Reverse (5=-ACTCTTGTGGTTGTGGAGCA-3=) and the probe (5=-AAGTTGCTCCAGTTGAACCAACTCCA-3=), 5= labeled with 6-carboxyfluorescein and the 3= labeled with 6-carboxytetramethylrhodamine, were used. A quantification standard, pGEM-ompA, was constructed by inserting the ompA gene from the ATCC 19606 strain...

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Checklist for optimization and validation of real‐time PCR assays

Cited by 159 publications

References 68 publications

Aichivirus in Children with Diarrhea in Northern Italy

Aichivirus in Children with Diarrhea in Northern Italy

Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR

Quantitative Real-Time PCR for Detection of Acinetobacter baumannii Colonization in the Hospital Environment

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