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Development of a rapid recombinase polymerase amplification assay for the detection of Streptococcus pneumoniae in whole blood.

Clancy E, Higgins O, Forrest MS, Boo TW, Cormican M, Barry T, Piepenburg O, Smith TJ - BMC Infect. Dis. (2015)

Bottom Line: The introduction of multivalent vaccines has coincided with a dramatic decrease in the number of pneumococcal-related deaths.In spite of this, at a global level, pneumococcal infection remains an important cause of death among children under 5 years of age and in adults 65 years of age or older.The inhibitory effect of human genomic DNA on amplification was investigated.

View Article: PubMed Central - PubMed

Affiliation: Molecular Diagnostics Research Group, School of Natural Sciences, National University of Ireland, Galway, Ireland. eoin.clancy@nuigalway.ie.

ABSTRACT

Background: Streptococcus pneumoniae is an important cause of microbial disease in humans. The introduction of multivalent vaccines has coincided with a dramatic decrease in the number of pneumococcal-related deaths. In spite of this, at a global level, pneumococcal infection remains an important cause of death among children under 5 years of age and in adults 65 years of age or older. In order to properly manage patients and control the spread of infection, a rapid and highly sensitive diagnostic method is needed for routine implementation, especially in resource-limited regions where pneumococcal disease is most prevalent.

Methods: Using the gene encoding leader peptidase A as a molecular diagnostics target, a real-time RPA assay was designed and optimised for the detection of S. pneumoniae in whole blood. The performance of the assay was compared to real-time PCR in terms of its analytical limit of detection and specificity. The inhibitory effect of human genomic DNA on amplification was investigated. The potential clinical utility of the assay was investigated using a small number of clinical samples.

Results: The RPA assay has a limit of detection equivalent to PCR (4.0 and 5.1 genome equivalents per reaction, respectively) and was capable of detecting the equivalent of <1 colony forming unit of S. pneumoniae when spiked into human whole blood. The RPA assay was 100 % inclusive (38/38 laboratory reference strains and 19/19 invasive clinical isolates) and 100 % exclusive; differentiating strains of S. pneumoniae species from other viridans group streptococci, including S. pseudopneumoniae. When applied to the analysis of a small number (n = 11) of clinical samples (blood culture positive for S. pneumoniae), the RPA assay was demonstrated to be both rapid and sensitive.

Conclusions: The RPA assay developed in this work is shown to be as sensitive and as specific as PCR. In terms of reaction kinetics, the RPA assay is shown to exceed those of the PCR, with the RPA running to completion in 20 minutes and capable generating a positive signal in as little as 6 minutes. This work represents a potentially suitable assay for application in point-of-care settings.

No MeSH data available.


Related in: MedlinePlus

RPA assay robustness. The graphs show the fluorescence intensity obtained from the amplification of 50, 20 and 4 genome equivalents (GE) of S. pneumoniae (type strain; DSM20566), a in the absence of background human genomic DNA, b in the presence of 200 ng background human genomic DNA and c in the presence of 400 ng background human genomic DNA. Also shown in each graph is the fluorescence intensity obtained from the amplification of 100 GE (positive control, PC) of S. pneumoniae (type strain; DSM20566) and a no template control reaction (NTC)
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Fig3: RPA assay robustness. The graphs show the fluorescence intensity obtained from the amplification of 50, 20 and 4 genome equivalents (GE) of S. pneumoniae (type strain; DSM20566), a in the absence of background human genomic DNA, b in the presence of 200 ng background human genomic DNA and c in the presence of 400 ng background human genomic DNA. Also shown in each graph is the fluorescence intensity obtained from the amplification of 100 GE (positive control, PC) of S. pneumoniae (type strain; DSM20566) and a no template control reaction (NTC)

Mentions: The degree to which the RPA reaction was inhibited by the presence of human DNA was concentration dependant, varying from no significant inhibition (100 ng; data not shown) to substantial inhibition in the presence of 400 ng human DNA (the highest tested). Whilst inhibited, when spiked with 200 ng or 400 ng of human DNA, 50, 20 and 4 S. pneumoniae GE were detected, all producing signals above that of the negative control reaction (Fig. 3). When the PCR reaction was spiked with human DNA (100, 200 or 400 ng/reaction), there was no discernible shift in threshold-cycle between spiked and non-spiked reactions, indicating that the PCR was not inhibited by the presence of human DNA (data not shown).Fig. 3


Development of a rapid recombinase polymerase amplification assay for the detection of Streptococcus pneumoniae in whole blood.

Clancy E, Higgins O, Forrest MS, Boo TW, Cormican M, Barry T, Piepenburg O, Smith TJ - BMC Infect. Dis. (2015)

RPA assay robustness. The graphs show the fluorescence intensity obtained from the amplification of 50, 20 and 4 genome equivalents (GE) of S. pneumoniae (type strain; DSM20566), a in the absence of background human genomic DNA, b in the presence of 200 ng background human genomic DNA and c in the presence of 400 ng background human genomic DNA. Also shown in each graph is the fluorescence intensity obtained from the amplification of 100 GE (positive control, PC) of S. pneumoniae (type strain; DSM20566) and a no template control reaction (NTC)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4625855&req=5

Fig3: RPA assay robustness. The graphs show the fluorescence intensity obtained from the amplification of 50, 20 and 4 genome equivalents (GE) of S. pneumoniae (type strain; DSM20566), a in the absence of background human genomic DNA, b in the presence of 200 ng background human genomic DNA and c in the presence of 400 ng background human genomic DNA. Also shown in each graph is the fluorescence intensity obtained from the amplification of 100 GE (positive control, PC) of S. pneumoniae (type strain; DSM20566) and a no template control reaction (NTC)
Mentions: The degree to which the RPA reaction was inhibited by the presence of human DNA was concentration dependant, varying from no significant inhibition (100 ng; data not shown) to substantial inhibition in the presence of 400 ng human DNA (the highest tested). Whilst inhibited, when spiked with 200 ng or 400 ng of human DNA, 50, 20 and 4 S. pneumoniae GE were detected, all producing signals above that of the negative control reaction (Fig. 3). When the PCR reaction was spiked with human DNA (100, 200 or 400 ng/reaction), there was no discernible shift in threshold-cycle between spiked and non-spiked reactions, indicating that the PCR was not inhibited by the presence of human DNA (data not shown).Fig. 3

Bottom Line: The introduction of multivalent vaccines has coincided with a dramatic decrease in the number of pneumococcal-related deaths.In spite of this, at a global level, pneumococcal infection remains an important cause of death among children under 5 years of age and in adults 65 years of age or older.The inhibitory effect of human genomic DNA on amplification was investigated.

View Article: PubMed Central - PubMed

Affiliation: Molecular Diagnostics Research Group, School of Natural Sciences, National University of Ireland, Galway, Ireland. eoin.clancy@nuigalway.ie.

ABSTRACT

Background: Streptococcus pneumoniae is an important cause of microbial disease in humans. The introduction of multivalent vaccines has coincided with a dramatic decrease in the number of pneumococcal-related deaths. In spite of this, at a global level, pneumococcal infection remains an important cause of death among children under 5 years of age and in adults 65 years of age or older. In order to properly manage patients and control the spread of infection, a rapid and highly sensitive diagnostic method is needed for routine implementation, especially in resource-limited regions where pneumococcal disease is most prevalent.

Methods: Using the gene encoding leader peptidase A as a molecular diagnostics target, a real-time RPA assay was designed and optimised for the detection of S. pneumoniae in whole blood. The performance of the assay was compared to real-time PCR in terms of its analytical limit of detection and specificity. The inhibitory effect of human genomic DNA on amplification was investigated. The potential clinical utility of the assay was investigated using a small number of clinical samples.

Results: The RPA assay has a limit of detection equivalent to PCR (4.0 and 5.1 genome equivalents per reaction, respectively) and was capable of detecting the equivalent of <1 colony forming unit of S. pneumoniae when spiked into human whole blood. The RPA assay was 100 % inclusive (38/38 laboratory reference strains and 19/19 invasive clinical isolates) and 100 % exclusive; differentiating strains of S. pneumoniae species from other viridans group streptococci, including S. pseudopneumoniae. When applied to the analysis of a small number (n = 11) of clinical samples (blood culture positive for S. pneumoniae), the RPA assay was demonstrated to be both rapid and sensitive.

Conclusions: The RPA assay developed in this work is shown to be as sensitive and as specific as PCR. In terms of reaction kinetics, the RPA assay is shown to exceed those of the PCR, with the RPA running to completion in 20 minutes and capable generating a positive signal in as little as 6 minutes. This work represents a potentially suitable assay for application in point-of-care settings.

No MeSH data available.


Related in: MedlinePlus