COMPARATIVE STUDY OF SPECIFICITY AND SENSITIVITY FOR IHHNV DETECTION BETWEEN REAL-TIME PCR AND LAMP METHODS

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

Download Full-text

A comparative study of DNA extraction methods to be used in real-time PCR based quantification of ochratoxin A-producing molds in food products

Food Control ◽

10.1016/j.foodcont.2011.12.010 ◽

2012 ◽

Vol 25 (2) ◽

pp. 666-672 ◽

Cited By ~ 23

Author(s):

Alicia Rodríguez ◽

Mar Rodríguez ◽

M. Isabel Luque ◽

Annemarie F. Justesen ◽

Juan J. Córdoba

Keyword(s):

Comparative Study ◽

Real Time ◽

Ochratoxin A ◽

Dna Extraction ◽

Real Time Pcr ◽

Food Products ◽

Extraction Methods ◽

Dna Extraction Methods

Download Full-text

Detection and identification of Acanthamoeba and other non-viral causes of infectious keratitis in corneal scrapings by real-time PCR and next-generation sequencing-based 16S-18S gene analysis

Journal of Clinical Microbiology ◽

10.1128/jcm.02224-20 ◽

2020 ◽

Author(s):

Dennis Back Holmgaard ◽

Celine Barnadas ◽

Seyed Hossein Mirbarati ◽

Lee O’Brien Andersen ◽

Henrik Vedel Nielsen ◽

...

Keyword(s):

Next Generation Sequencing ◽

Real Time ◽

Real Time Pcr ◽

Clinical Relevance ◽

Bacterial Species ◽

Screening Method ◽

Infectious Keratitis ◽

Next Generation ◽

Specificity And Sensitivity ◽

Generation Sequencing

Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive using specific real-time PCR, 21 of which were positive using the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). Conclusively, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotype was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting non-viral causes of IK, including Acanthamoeba.

Download Full-text

Real-time PCR: A rapid and sensitive method for diagnosis of dermatophyte induced onychomycosis, a comparative study

Alexandria Journal of Medicine ◽

10.1016/j.ajme.2015.05.001 ◽

2016 ◽

Vol 52 (1) ◽

pp. 83-90 ◽

Cited By ~ 4

Author(s):

Sherin M. Emam ◽

Osama H. Abd El-salam

Keyword(s):

Comparative Study ◽

Real Time ◽

Real Time Pcr ◽

Sensitive Method

Download Full-text

A Comparative Study of Six Trichinella Species by Real-time PCR Assay

10.7546/grabs2018.1.08 ◽

2018 ◽

Author(s):

Valeria Dilcheva ◽

Ivelin Vladov ◽

Svetlozara Petkova

Keyword(s):

Comparative Study ◽

Real Time ◽

Real Time Pcr ◽

Pcr Assay

Download Full-text

Comparison of real-time PCR protocols in detection and quantification of fruit tree 16SrX group phytoplasmas

Genetika ◽

10.2298/gensr1602629k ◽

2016 ◽

Vol 48 (2) ◽

pp. 629-642 ◽

Cited By ~ 1

Author(s):

Tomas Kiss ◽

Tomas Necas ◽

Jana Necasova

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Nested Pcr ◽

Fruit Tree ◽

Amplification Efficiency ◽

Specificity And Sensitivity ◽

Intercalating Dye ◽

Detection Characteristics ◽

Detection And Quantification

In this work, two real-time PCR protocols based on intercalating dye and two on hydrolysis probes were tested using field collected fruit tree samples infected by 16SrX group (AP, PD and ESFY) phytoplasmas. Specificity and sensitivity of protocols and amplification efficiency were the main testing parameters. Results of real-time PCR protocols were compared to nested PCR. All real-time PCR protocols confirmed their specificity of detection. All real-time PCR protocols were 10-100 times more sensitive than nested PCR. Afterall real-time PCR protocols based on hydrolysis probes were 10 times more sensitive than protocols based on intercalating dyes. Among protocols based on hydrolysis probes, slightly better detection characteristics were shown by protocol by CHRISTENSEN et al. (2004).

Download Full-text