Novel locked nucleic acid (LNA)-based probe for the rapid identification of Chlamydia suisusing real-time PCR

We developed a novel real-time PCR assay that simultaneously evaluates eleven major nucleos(t)ide antiviral (NA) drug-resistance mutations (mt) in chronic hepatitis B patients (CHB), including L180M, M204I/V, and V207M (lamivudine (LMV) resistance), N/H238A/T (Adefovir (ADF) resistance) which are circulating in Vietnam; T184G/L, S202I, M250V (entecavir (ETV) resistance), and A194T (tenofovir resistance) which have been recently reported in several studies across the globe. We detected drug-resistant mt in HBV samples using our predesigned panel of allele-specific locked-nucleic acid (LNA) probes. Our assay had a high sensitivity of 5% in a low HBV DNA population of ≥5 ×10 3 IU/mL and was validated in a cohort of 130 treatment-naive children and 98 NA-experienced adults with CHB. Single-point mt for LMV- and ADF-resistance were detected in 57.7% and 54.1% of the child and adult samples, respectively, with rtV207M (42.3%, children; 36.7%, adults) and rtN238T/A (15.4%, children; 16.3, adults) being the most frequent mt in these populations. Multiple-point mt including rtL180M-rtM204V- rtN238A and rtL180M-rtM204I were only identified in two children, resulting in LMV-ADF-resistance and reduced ETV susceptibility. In conclusion, this assay accurately identified the mt profile of children (98.4%) and adults (91.2%) with CHB, which is comparable to established methods. This fast and sensitive screening method can be used for the detection of major NA-resistant mt circulating in developing countries, as well as providing a model for the development of similar mt-detection assays, especially for use in non-hospitalised patients who need their results within half a day before starting treatment.

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Development of a real-time PCR assay using Locked Nucleic Acid probe for the detection of octopine, nopaline, and vitopine strains of tumorigenic Allorhizobium vitis in grapevines

Crop Protection ◽

10.1016/j.cropro.2021.105620 ◽

2021 ◽

pp. 105620

Author(s):

Ali Turgut ◽

Hüseyin BASIM

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Locked Nucleic Acid ◽

Pcr Assay ◽

Allorhizobium Vitis ◽

Locked Nucleic Acid Probe

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Multiplex TaqMan locked nucleic acid real-time PCR for the differential identification of various meat and meat products

Meat Science ◽

10.1016/j.meatsci.2017.11.003 ◽

2018 ◽

Vol 137 ◽

pp. 41-46 ◽

Cited By ~ 22

Author(s):

Rusu Xu ◽

Shuang Wei ◽

Guangbiao Zhou ◽

Jiao Ren ◽

Zhongyong Liu ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Meat Products ◽

Locked Nucleic Acid ◽

Differential Identification

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Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR

Nucleic Acids Research ◽

10.1093/nar/gnh046 ◽

2004 ◽

Vol 32 (6) ◽

pp. e55-e55 ◽

Cited By ~ 106

Author(s):

M. P. Johnson

Keyword(s):

Single Nucleotide Polymorphism ◽

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Locked Nucleic Acid ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Genotype Analysis

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Library of Prefabricated Locked Nucleic Acid Hydrolysis Probes Facilitates Rapid Development of Reverse-Transcription Quantitative Real-Time PCR Assays for Detection of Novel Influenza A/H1N1/09 Virus

Clinical Chemistry ◽

10.1373/clinchem.2009.136192 ◽

2009 ◽

Vol 55 (12) ◽

pp. 2218-2222 ◽

Cited By ~ 16

Author(s):

Jürgen J Wenzel ◽

Heiko Walch ◽

Markus Bollwein ◽

Hans Helmut Niller ◽

Waltraud Ankenbauer ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Reverse Transcription ◽

Real Time Pcr ◽

Influenza A ◽

Rapid Development ◽

Locked Nucleic Acid ◽

Quantitative Real Time Pcr ◽

Influenza A H1n1 ◽

Novel Influenza A

Abstract Background: The emergence of a novel pandemic human strain of influenza A (H1N1/09) has clearly demonstrated the need for flexible tools enabling the rapid development of new diagnostic methods. Methods: We designed a set of reverse-transcription quantitative real-time PCR (RT-qPCR) assays based on the Universal ProbeLibrary (UPL)—a collection of 165 presynthesized, fluorescence-labeled locked nucleic acid (LNA) hydrolysis probes—specifically to detect the novel influenza A virus. We evaluated candidate primer/UPL-probe pairs with 28 novel influenza A/H1N1/09 patient samples of European and Mexican origin. Results: Of 14 assays in the hemagglutinin (HA) and neuraminidase (NA) genes, 12 detected viral nucleic acids from diluted patient samples without need for further optimization. We characterized the diagnostic specificity of the 2 best-performing assays with a set of samples comprising various influenza virus strains of human and animal origin that showed no cross-reactivity. The diagnostic sensitivity of these 2 primer/probe combinations was in the range of 100–1000 genomic copies/mL. In comparison to a reference assay recommended by the German health authorities, the analytical sensitivities and specificities of the assays were equivalent. Conclusions: Facing the emergence of novel influenza A/H1N1/09, we were able to develop, within 2 days, a set of sensitive and specific RT-qPCR assays for the laboratory diagnosis of suspected cases. H1N1/09 served as a model to show the feasibility of the UPL approach for the expedited development of new diagnostic assays to detect emerging pathogens.

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