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Use of sensitive, broad-spectrum molecular assays and human airway epithelium cultures for detection of respiratory pathogens.

Pyrc K, Stożek K, Wojcik K, Gawron K, Zeglen S, Karolak W, Wojarski J, Ochman M, Hubalewska-Mazgaj M, Bochenek G, Sanak M, Zembala M, Szczeklik A, Potempa J - PLoS ONE (2012)

Bottom Line: Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ~76% of cases.This additional step resulted in the detection of pathogens in all samples tested.Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Department, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. k.a.pyrc@uj.edu.pl

ABSTRACT
Rapid and accurate detection and identification of viruses causing respiratory tract infections is important for patient care and disease control. Despite the fact that several assays are available, identification of an etiological agent is not possible in ~30% of patients suffering from respiratory tract diseases. Therefore, the aim of the current study was to develop a diagnostic set for the detection of respiratory viruses with sensitivity as low as 1-10 copies per reaction. Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ~76% of cases. To improve assay performance and facilitate the identification of novel species or emerging strains, cultures of fully differentiated human airway epithelium were used to pre-amplify infectious viruses. This additional step resulted in the detection of pathogens in all samples tested. Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used.

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Related in: MedlinePlus

Specificity of the developed assays.All primer pairs (names on top of the figure) were used to amplify all virus stocks included in the study (names on the left side of each panel). M: size marker (GeneRuler 50–1000 bp DNA; Fermentas); CoV_1: HCoV-NL63, CoV_2: HCoV-HKU1; RSV: respiratory syncytial virus; IAV: influenza A virus; PIV: parainfluenza virus type 1, 2 or 3; BoV: bocavirus; AdV: adenovirus type 4; IBV: influenza B virus; hMPV: human metapneumovirus; EV: echovirus 9. Analysis was performed on 1.5% agarose gel.
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pone-0032582-g004: Specificity of the developed assays.All primer pairs (names on top of the figure) were used to amplify all virus stocks included in the study (names on the left side of each panel). M: size marker (GeneRuler 50–1000 bp DNA; Fermentas); CoV_1: HCoV-NL63, CoV_2: HCoV-HKU1; RSV: respiratory syncytial virus; IAV: influenza A virus; PIV: parainfluenza virus type 1, 2 or 3; BoV: bocavirus; AdV: adenovirus type 4; IBV: influenza B virus; hMPV: human metapneumovirus; EV: echovirus 9. Analysis was performed on 1.5% agarose gel.

Mentions: To determine whether the newly designed assays were specific for the selected pathogen, all primer pairs were tested for cross-reactivity against all other respiratory pathogens included in the study. Briefly, RNA or DNA was isolated from concentrated cell culture samples or clinical specimens and subjected to reverse transcription, where appropriate. The resulting cDNA was used as an input for nested PCR using all the primer sets shown in Table 1. No cross-reactivity was detected in any of the assays, confirming high specificity of the PCR reactions (Figure 4).


Use of sensitive, broad-spectrum molecular assays and human airway epithelium cultures for detection of respiratory pathogens.

Pyrc K, Stożek K, Wojcik K, Gawron K, Zeglen S, Karolak W, Wojarski J, Ochman M, Hubalewska-Mazgaj M, Bochenek G, Sanak M, Zembala M, Szczeklik A, Potempa J - PLoS ONE (2012)

Specificity of the developed assays.All primer pairs (names on top of the figure) were used to amplify all virus stocks included in the study (names on the left side of each panel). M: size marker (GeneRuler 50–1000 bp DNA; Fermentas); CoV_1: HCoV-NL63, CoV_2: HCoV-HKU1; RSV: respiratory syncytial virus; IAV: influenza A virus; PIV: parainfluenza virus type 1, 2 or 3; BoV: bocavirus; AdV: adenovirus type 4; IBV: influenza B virus; hMPV: human metapneumovirus; EV: echovirus 9. Analysis was performed on 1.5% agarose gel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293820&req=5

pone-0032582-g004: Specificity of the developed assays.All primer pairs (names on top of the figure) were used to amplify all virus stocks included in the study (names on the left side of each panel). M: size marker (GeneRuler 50–1000 bp DNA; Fermentas); CoV_1: HCoV-NL63, CoV_2: HCoV-HKU1; RSV: respiratory syncytial virus; IAV: influenza A virus; PIV: parainfluenza virus type 1, 2 or 3; BoV: bocavirus; AdV: adenovirus type 4; IBV: influenza B virus; hMPV: human metapneumovirus; EV: echovirus 9. Analysis was performed on 1.5% agarose gel.
Mentions: To determine whether the newly designed assays were specific for the selected pathogen, all primer pairs were tested for cross-reactivity against all other respiratory pathogens included in the study. Briefly, RNA or DNA was isolated from concentrated cell culture samples or clinical specimens and subjected to reverse transcription, where appropriate. The resulting cDNA was used as an input for nested PCR using all the primer sets shown in Table 1. No cross-reactivity was detected in any of the assays, confirming high specificity of the PCR reactions (Figure 4).

Bottom Line: Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ~76% of cases.This additional step resulted in the detection of pathogens in all samples tested.Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Department, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. k.a.pyrc@uj.edu.pl

ABSTRACT
Rapid and accurate detection and identification of viruses causing respiratory tract infections is important for patient care and disease control. Despite the fact that several assays are available, identification of an etiological agent is not possible in ~30% of patients suffering from respiratory tract diseases. Therefore, the aim of the current study was to develop a diagnostic set for the detection of respiratory viruses with sensitivity as low as 1-10 copies per reaction. Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ~76% of cases. To improve assay performance and facilitate the identification of novel species or emerging strains, cultures of fully differentiated human airway epithelium were used to pre-amplify infectious viruses. This additional step resulted in the detection of pathogens in all samples tested. Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used.

Show MeSH
Related in: MedlinePlus