A multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies

Molecular epidemiology is one of the most rapidly developing research area. In the light of past and modern technologies it has gained number of typing methods based on molecular biology techniques. In this report, the subspecies differentiation of Francisella tularensis and genotyping of strains isolated in Poland and other geographic locations were investigated using real-time PCR and multispacer typing (MST) methods respectively. In total, 49 strains of F. tularensis included 15 strains from Poland, for subspecies differentiation the real-time PCR method was applied. For molecular typing using MST method four intergenic spacers (IS) were sequenced and compared with those previously described and deposited in GenBank (NCBI). Phylogenetic testing was performed using with the UPGMA model using MEGA6 software. The real-time PCR enables to distinguish five strains belonged to type A and 44 to type B among deposited F. tularensis strains. The MST revealed previously described genotypes, as well as 23 new genotypes were detected. The use of real-time PCR and MST method are valuable in the analysis of F. tularensis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrate convenient molecular tools (real-time PCR and multispacer typing) for Francisella tularensis detection, differentiation and genotyping which can be applied for molecular epidemiological studies and providing useful information for scientific research and during natural tularaemia outbreaks.

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“…; Gunnell et al . ; Zasada et al . ) as well as subspecies differentiation (Molins‐Schneekloth et al .…”

Section: Resultsmentioning

confidence: 98%

Subspecies differentiation and genotyping ofFrancisella tularensisstrains isolated from clinical and environmental samples

Cieślik

Knap

Piechowicz

et al. 2018

Lett Appl Microbiol

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“…A similar strategy was carried out for the identification of Gentiana macrophylla 42 . Furthermore, the application has also been successfully used in the detection of a bacterial pathogen and HIV virological failure 43 44 .…”

Section: Discussionmentioning

confidence: 99%

An integrated system for identifying the hidden assassins in traditional medicines containing aristolochic acids

Sun

Wang

et al. 2015

Sci Rep

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Traditional herbal medicines adulterated and contaminated with plant materials from the Aristolochiaceae family, which contain aristolochic acids (AAs), cause aristolochic acid nephropathy. Approximately 256 traditional Chinese patent medicines, containing Aristolochiaceous materials, are still being sold in Chinese markets today. In order to protect consumers from health risks due to AAs, the hidden assassins, efficient methods to differentiate Aristolochiaceous herbs from their putative substitutes need to be established. In this study, 158 Aristolochiaceous samples representing 46 species and four genera as well as 131 non-Aristolochiaceous samples representing 33 species, 20 genera and 12 families were analyzed using DNA barcodes based on the ITS2 and psbA-trnH sequences. Aristolochiaceous materials and their non-Aristolochiaceous substitutes were successfully identified using BLAST1, the nearest distance method and the neighbor-joining (NJ) tree. In addition, based on sequence information of ITS2, we developed a Real-Time PCR assay which successfully identified herbal material from the Aristolochiaceae family. Using Ultra High Performance Liquid Chromatography-Mass Spectrometer (UHPLC-HR-MS), we demonstrated that most representatives from the Aristolochiaceae family contain toxic AAs. Therefore, integrated DNA barcodes, Real-Time PCR assays using TaqMan probes and UHPLC-HR-MS system provides an efficient and reliable authentication system to protect consumers from health risks due to the hidden assassins (AAs).

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“…17 , 38 , 40 There is potential for available laboratory kits to be re-engineered for NPT/POC testing, including kits for viral haemorrhagic pathogens 41 , 42 , 43 , 44 , arboviral diseases, 45 , 46 , 47 , 48 respiratory pathogens 49 , 50 , 51 and biothreat pathogens (including Disease X). 39 , 52 , 53 …”

Section: Molecular Diagnostics For Epidemic Preparednessmentioning

confidence: 99%

An open-source molecular diagnostic platform approach for outbreak and epidemic preparedness

Emperador

Mazzola²,

Kelly-Cirino

2020

Afr. J. Lab. Med.

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Background: Diagnostic development for outbreak pathogens has typically followed a disease-specific reactive rather than proactive response. Given the diversity of outbreak pathogens, particularly those prioritised by the World Health Organization Research and Development Blueprint, a more flexible and proactive approach to epidemic preparedness is needed to expand access to critical molecular diagnostic tests in peripheral and resource-constrained deployment settings.Objective: New and more sustainable directives are needed to spur the development of high-quality products, particularly for epidemics more often found in low- and middle-income countries. To leverage and de-risk the development process, we present the benefits and challenges of an open-source business model for co-development of molecular diagnostic tests for decentralised settings.Methods: We identify key outbreak pathogens that are available only for testing in high infrastructure laboratories and compare in-country installed base platforms that could be leveraged for menu expansion. Key strengths and challenges for development are highlighted for both platform and assay developers, with discussion of how to leverage and de-risk the process through an open-source development model.Results: Depending on the specific partner strengths, options for partnership roles are presented. The proposed open-source business model addresses the particular challenges in the detection of outbreak- and epidemic-prone pathogens in low- and middle-income countries, reduces development and deployment risks to support outbreak response, strengthens diagnostic capacity and creates a viable market for product developers.Conclusion: We hope this model for a collaborative and open-source approach for molecular diagnostics serves to encourage stakeholders to consider co-development partnerships to improve outbreak preparedness and epidemic/pandemic response.

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A multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies

Cited by 15 publications

References 31 publications

Subspecies differentiation and genotyping ofFrancisella tularensisstrains isolated from clinical and environmental samples

Subspecies differentiation and genotyping ofFrancisella tularensisstrains isolated from clinical and environmental samples

An integrated system for identifying the hidden assassins in traditional medicines containing aristolochic acids

An open-source molecular diagnostic platform approach for outbreak and epidemic preparedness

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