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Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses
Published on: November 10, 2023
Standardisation and evaluation of a quantitative multiplex real-time PCR assay for the rapid identification of
Feroze A Ganaie1, Vandana Govindan1, K L Ravi Kumar1
1Department of Microbiology, Kempegowda Institute of Medical Sciences, Hospital and Research Centre, K.R Road, V.V Puram, Bangalore, 560004 India.
Abstract:
Rapid diagnosis of Streptococcus pneumoniae can play a significant role in decreasing morbidity and mortality of infection. The accurate diagnosis of pneumococcal disease is hampered by the difficulties in growing the isolates from clinical specimens and also by misidentification. Molecular methods have gained popularity as they offer improvement in the detection of causative pathogens with speed and ease. The present study aims at validating and standardising the use of 4 oligonucleotide primer-probe sets (pneumolysin [ply], autolysin [lytA], pneumococcal surface adhesion A [psaA] and Spn9802 [DNA fragment]) in a single-reaction mixture for the detection and discrimination of S. pneumoniae. Here, we validate a quantitative multiplex real-time PCR (qmPCR) assay with a panel consisting of 43 S. pneumoniae and 29 non-pneumococcal isolates, 20 culture positive, 26 culture negative and 30 spiked serum samples. A standard curve was obtained using S. pneumoniae ATCC 49619 strain and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous internal control. The experiment showed high sensitivity with lower limit of detection equivalent to 4 genome copies/µl. The efficiency of the reaction was 100% for ply, lytA, Spn9802 and 97% for psaA. The test showed sensitivity and specificity of 100% with culture isolates and serum specimens. This study demonstrates that qmPCR analysis of sera using 4 oligonucleotide primers appears to be an appropriate method for the genotypic identification of S. pneumoniae infection.
Insights
Rapid diagnosis of Streptococcus pneumoniae infections is crucial. A new quantitative multiplex real-time PCR assay accurately detects S. pneumoniae in clinical samples, improving patient outcomes.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Accurate and rapid diagnosis of Streptococcus pneumoniae infections is critical for reducing morbidity and mortality.
- Traditional diagnostic methods face challenges with isolate cultivation and potential misidentification.
- Molecular techniques offer enhanced speed and accuracy in pathogen detection.
Purpose of the Study:
- To validate and standardize a quantitative multiplex real-time PCR (qmPCR) assay for detecting and differentiating Streptococcus pneumoniae.
- To assess the assay's performance using multiple primer-probe sets in a single reaction.
- To evaluate the qmPCR assay's utility in clinical specimens, including serum samples.
Main Methods:
- Development and validation of a qmPCR assay using four primer-probe sets targeting pneumolysin (ply), autolysin (lytA), pneumococcal surface adhesion A (psaA), and Spn9802.
- Testing the assay with a panel of 43 S. pneumoniae isolates, 29 non-pneumococcal isolates, culture-positive and negative samples, and spiked serum samples.
- Utilizing a standard curve from S. pneumoniae ATCC 49619 and GAPDH as an internal control.
Main Results:
- The qmPCR assay demonstrated high sensitivity with a lower limit of detection of 4 genome copies/µl.
- Reaction efficiencies were high, ranging from 97% to 100% for the targeted genes.
- The assay achieved 100% sensitivity and specificity when tested with culture isolates and serum specimens.
Conclusions:
- The validated qmPCR assay is a highly sensitive and specific method for the rapid genotypic identification of Streptococcus pneumoniae infections.
- This molecular approach overcomes limitations of traditional culture-based methods.
- The qmPCR assay shows significant potential for routine clinical diagnostics of pneumococcal disease.
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