Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model

Monteiro, Lurdes; Bonnemaison, Dominique; Vekris, Antoine; Petry, Klaus G.; Bonnet, Jacques; Vidal, Rui; Cabrita, José; Mégraud, Françis

doi:10.1128/jcm.35.4.995-998.1997

Cited by 450 publications

(221 citation statements)

References 17 publications

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“…The use of fecal and nasal swab samples as the most frequent matrices for detection of BCoV in diseased animals constitutes one of the significant problems that may hinder the performance of RT-PCR assays. Considerable existence of the bile salts and the complex polysaccharides in these matrices can either block the enzyme activity or reduce the Magnesium salt availability [25]. Therefore, monitoring PCR inhibitors in the utilized samples is very important to avoid false negative results.…”

Section: Discussionmentioning

confidence: 99%

Development of a SYBR Green I based real-time RT-PCR assay for detection and quantification of bovine coronavirus

Amer

Almajhdi

2011

Molecular and Cellular Probes

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A novel two-step, SYBR Green I based real-time RT-PCR assay was developed for detection and quantification of BCoV using ABI PRISM 7500 sequence detection system. The assay was carried out using two sets of primers designed to amplify highly conserved sequences of the nucleocapsid gene of BCoV and the internal control, bovine glyceraldehyde-3-phosphate dehydrogenase, RNA. Specific identification of both targets was elucidated by melt curve analysis, in which the BCoV amplified product generated a melt peak at 78.35 ± 0.26 °C and the internal control RNA at 82.54 ± 0.32 °C. The assay was highly specific since all negative controls and other viruses of clinical and structural relevance failed to develop any positive results. The detection limit of the reaction was 10(3) plasmid copies and 1.17 × 10(-3) TCID(50) of the tissue culture propagated virus. Standard deviation and coefficient of variation was low for both intra-assay and inter-assay variability. The assay performance on field samples was evaluated on 103 (68 fecal and 35 nasal) swab specimens and compared with the conventional RT-PCR assay. The results of both assays matched for the diagnosis of 65 fecal and 33 nasal samples. However, three fecal and two nasal samples tested negative in gel-based assay were positive for the real-time RT-PCR. The robustness and a high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and in BCoV research.

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Section: Discussionmentioning

confidence: 99%

Development of a SYBR Green I based real-time RT-PCR assay for detection and quantification of bovine coronavirus

Amer

Almajhdi

2011

Molecular and Cellular Probes

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“…The combination of phenol/chloroform/isoamyl alcohol and silica/guanidinium isothiocyanate methods was performed according to Barreiros et al (2004) with slight modifications. Faecal samples remain the most difficult specimens for nucleic acid extraction and amplification due the presence of inhibitors (Monteiro et al, 1997). Guanidinium isothiocyanate alone or in combination with phenol-chloroform inactivates ribonucleases and therefore improves the stability of the extracted RNA genome (Romero, 1999).…”

Section: Discussionmentioning

confidence: 99%

“…Besides, there are no comparative studies on the ability of different primer sets to detect BCoV in clinical samples. Also, faeces remains the most difficult clinical sample for nucleic acid extraction and amplification due to the presence of PCR inhibitors that may yield false negative results (Monteiro et al, 1997). The use of an internal control in the PCR reaction has been most commonly applied to monitor and evaluate these failures, but this approach has not been used in assays for BCoV detection (Beld et al, 2004;Dingle et al, 2004;Hoorfar et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Improved detection of bovine coronavirus N gene in faeces of calves infected naturally by a semi-nested PCR assay and an internal control

Takiuchi

Stipp

Alfieri

2006

Journal of Virological Methods

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Bovine coronavirus (BCoV), a positive sense single-stranded RNA virus, is an important causative agent of neonatal diarrhoea in calves from beef and dairy cattle worldwide. The routine detection and diagnosis of BCoV have been mainly dependent on assays with low sensitivity. The aim of the present study was to develop and evaluate a semi-nested PCR (SN-PCR) to amplify a 251bp fragment of BCoV N gene from fresh (n=25) and frozen (n=25) diarrhoeic faecal samples of naturally infected calves. To improve detection of BCoV in faecal samples by the SN-PCR an internal control was developed, and the results were compared with a conventional RT-PCR assay. The rates of positive samples by SN-PCR and RT-PCR were 24% (12/50) and 8% (4/50), respectively (K=0.43). Only fresh samples were positive in RT-PCR while the SN-PCR detected BCoV in both fresh and frozen faecal samples. The sensitivity of SN-PCR was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) that was detected until 10(-7) dilution. The specificity of the amplicons was assessed by restriction fragment length polymorphism and sequence analysis. The inclusion of an internal control provides a way to detect assay inhibition in faecal samples and failure of nucleic acid extraction that allow reduction of the number of false-negative results.

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“…Most published microarray methods use amplification strategies prior to hybridization that are based on the polymerase chain reaction (Saiki et al, 1988;Mullis et al, 1986), a proven and sensitive technique. However, PCR is susceptible to numerous inhibitory compounds commonly present in clinical materials (Monteiro et al, 1997;Fredricks and Relman, 1998;Longo et al, 1990); fastidious sample preparation is thus required to obtain robust detection (Klein et al, 1997). In addition, some microorganisms potentially responsible for infections harbor specific envelopes or cell-walls which are either difficult to disrupt or further release inhibitors of enzymatic reactions.…”

Section: Sample Preparationmentioning

confidence: 99%