SPECIFIC DETECTION OF SARS-COV-2 B.1.1.529 (OMICRON) VARIANT BY FOUR RT-qPCR DIFFERENTIAL ASSAYS

Mapping Intimacies ◽

10.1101/2021.12.07.21267293 ◽

2021 ◽

Author(s):

ORAN ERSTER ◽

Adi Beth Din ◽

Hadar Asraf ◽

Virginia Levy ◽

Areej Kabat ◽

...

Keyword(s):

Rapid Identification ◽

Specific Detection ◽

First Line

In this report, we describe four RT-qPCR assays that enable rapid identification of the newly emerging SARS-COV-2 Omicron (B.1.1.529) variant of concern. The assays target Omicron characteristic mutations in the nsp6 (Orf1a), spike and nucleocapsid genes. We demonstrate that the assays are straightforward to assemble and perform, are amendable for multiplexing, and may be used as a reliable first-line tool to identify B.1.1.529 suspected samples. Importantly, this is a preliminary development report. Further validation and optimization of the assays described herein will be published hereafter.

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Rapid Identification, Differentiation, and Proposed New Taxonomic Classification of Bifidobacterium lactis

Applied and Environmental Microbiology ◽

10.1128/aem.68.12.6429-6434.2002 ◽

2002 ◽

Vol 68 (12) ◽

pp. 6429-6434 ◽

Cited By ~ 83

Author(s):

Marco Ventura ◽

Ralf Zink

Keyword(s):

Pcr Primers ◽

Rapid Identification ◽

Molecular Techniques ◽

Rrna Gene ◽

Specific Detection ◽

Bifidobacterium Lactis ◽

Bifidobacterium Animalis ◽

Subspecies Level ◽

Internally Transcribed Spacer

ABSTRACT Identification of Bifidobacterium lactis and Bifidobacterium animalis is problematic because of phenotypic and genetic homogeneities and has raised the question of whether they belong to one unique taxon. Analysis of the 16S-23S internally transcribed spacer region of B. lactis DSM10140T, B. animalis ATCC 25527T, and six potential B. lactis strains suggested two distinct clusters. Two specific 16S-23S spacer rRNA gene-targeted primers have been developed for specific detection of B. animalis. All of the molecular techniques used (B. lactis or B. animalis PCR primers, enterobacterial repetitive intergenic consensus PCR) demonstrated that B. lactis and B. animalis form two main groups and suggest a revision of the strains assigned to B. animalis. We propose that B. lactis should be separated from B. animalis at the subspecies level.

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Nested PCR for specific detection and rapid identification of human picornaviruses.

Journal of Clinical Microbiology ◽

10.1128/jcm.32.2.285-291.1994 ◽

1994 ◽

Vol 32 (2) ◽

pp. 285-291 ◽

Cited By ~ 59

Author(s):

U Kämmerer ◽

B Kunkel ◽

K Korn

Keyword(s):

Nested Pcr ◽

Rapid Identification ◽

Specific Detection

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Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay.

Journal of Clinical Microbiology ◽

10.1128/jcm.34.10.2619-2622.1996 ◽

1996 ◽

Vol 34 (10) ◽

pp. 2619-2622 ◽

Cited By ~ 188

Author(s):

C H Chiu ◽

J T Ou

Keyword(s):

Multiplex Pcr ◽

Virulence Genes ◽

Rapid Identification ◽

Broth Culture ◽

Specific Detection ◽

Enrichment Broth ◽

Salmonella Serovars

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Specific Detection and Rapid Identification of Human Enteroviruses by PCR Amplification

Methods in DNA Amplification ◽

10.1007/978-1-4615-2530-1_15 ◽

1994 ◽

pp. 127-134

Author(s):

U. Kämmerer ◽

B. Kunkel ◽

K. Korn

Keyword(s):

Pcr Amplification ◽

Rapid Identification ◽

Specific Detection

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Overcoming the Challenges of Pyrazinamide Susceptibility Testing in Clinical Mycobacterium tuberculosis isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02617-20 ◽

2021 ◽

Author(s):

Simone Mok ◽

Emma Roycroft ◽

Peter R Flanagan ◽

Lorraine Montgomery ◽

Emanuele Borroni ◽

...

Keyword(s):

Sanger Sequencing ◽

Susceptibility Testing ◽

Critical Concentration ◽

Drug Susceptibility ◽

Rapid Identification ◽

Drug Susceptibility Testing ◽

Whole Genome ◽

Testing Methods ◽

First Line ◽

Phenotypic Susceptibility

Pyrazinamide (PZA) is one of the first-line agents used for the treatment of tuberculosis. However, current phenotypic PZA susceptibility testing in the BACTEC MGIT 960 system is unreliable and false resistance is well documented. Rapid identification of resistance-associated mutations can confirm the phenotypic result. This study aimed to investigate the use of genotypic methods in combination with phenotypic susceptibility testing for confirmation of PZA resistant M. tuberculosis isolates. Sanger sequencing and/or whole genome sequencing were performed to detect mutations in pncA, rpsA, panD and clpC1. Isolates were screened for heteroresistance, and PZA susceptibility testing was performed in the BACTEC MGIT 960 system using a reduced inoculum to investigate false resistance. Overall, 40 phenotypically PZA resistant isolates were identified. Of these, PZA resistance was confirmed in 22/40 (55%) isolates by detecting mutations in pncA, rpsA and panD genes. 16/40 (40%) isolates were found to be susceptible using the reduced inoculum method (i.e. false resistance). No mutations were detected in two PZA resistant isolates. False resistance was observed in isolates with MICs close to the critical concentration. In particular, EAI strains (lineage 1) appeared to have an elevated MIC that is close to the critical concentration. While this study illustrates the complexity and challenges associated with PZA susceptibility testing of M. tuberculosis, we conclude that a combination of genotypic and phenotypic drug susceptibility testing methods is required for accurate detection of PZA resistance.

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Utility of Targeted, Amplicon-Based Deep Sequencing To Detect Resistance to First-Line Tuberculosis Drugs in Botswana

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00982-19 ◽

2019 ◽

Vol 63 (11) ◽

Author(s):

Qiao Wang ◽

Chawangwa Modongo ◽

Christopher Allender ◽

David M. Engelthaler ◽

Robin M. Warren ◽

...

Keyword(s):

Deep Sequencing ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Rapid Identification ◽

Drug Susceptibility Testing ◽

Lower Sensitivity ◽

First Line ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis ◽

High Concordance

ABSTRACT Multidrug-resistant tuberculosis (TB) is an alarming threat, and targeted deep sequencing (DS) may be an effective method for rapid identification of drug-resistant profiles, including detection of heteroresistance. We evaluated the sensitivity and specificity of targeted DS versus phenotypic drug susceptibility testing (pDST) among patients starting first-line anti-TB therapy in Botswana. Overall, we found high concordance between DS and pDST. Lower sensitivity of DS, which targets established high-confidence resistance variants, was observed for detecting isoniazid resistance among HIV-infected patients.

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Actinobaculum shaalii: a new uropathogen?

Microbiologia Medica ◽

10.4081/mm.2015.4801 ◽

2015 ◽

Vol 30 (1) ◽

Author(s):

Valentina Felice ◽

Massimiliano Scutellà ◽

Silvia Lombardi

Keyword(s):

Urinary Tract ◽

Urinary Tract Infections ◽

Diagnostic Procedures ◽

Rapid Identification ◽

First Line ◽

Emerging Pathogen ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Promising Tool ◽

Tract Infections

Background and Aims. Actinobaculum schaalii is a facultative anaerobic, Gram-positive rod-shaped species phylogenetically related to Actinomyces. A. schaalii is an emerging pathogen causing urinary tract infections (UTI) in both children and adults; although, as part of the human genitourinary tract flora, it is frequently overlooked or considered as a contaminant. While the phenotypic identification of A. schaalii is difficult, the recent Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight-mass spectrometry (MALDI TOF) technology could represent a promising tool for its identification. Materials and Methods. This is a retrospective study including all known cases (n=7) of A. schaalii infections occurred (between July 2013 and November 2013) at the Microbiology Laboratory of the A. Cardarelli Hospital, in Campobasso (Italy). Results. All the 7 A. schaalii collected strains, resulted in vitro susceptible to most of the drugs commonly used for urinary tract infections, but resistant to ciprofloxacin, a first-line antibiotic in the treatment of prostatitis. All isolates were susceptible to amoxicillin, amoxicillin-clavulanic, ampicillin-sulbactam, cefuroxime, gentamicin, piperacillin-tazobactam, vancomicin, tetracycline (no EUCAST breakpoints). All except two isolates were susceptible to cefotaxime; 3/7 and 5/7 strains were clindamicin and levofloxacin resistant, respectively. Conclusions. As most antibiotics empirically prescribed for UTI (mainly fluoroquinolones or trimethoprim/sulfamethoxazole) are not effective against A. schaalii, the appropriate onset of treatment was delayed by an average of 2.8 days. The implementation of the newer MALDI TOF technology in routine diagnostic procedures may allow a more reliable and rapid identification of A. schaalii in future.

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Use of an Enrichment Broth Cultivation-PCR Combination Assay for Rapid Diagnosis of Swine Erysipelas

Journal of Clinical Microbiology ◽

10.1128/jcm.36.1.86-89.1998 ◽

1998 ◽

Vol 36 (1) ◽

pp. 86-89 ◽

Cited By ~ 38

Author(s):

Yoshihiro Shimoji ◽

Yasuyuki Mori ◽

Koji Hyakutake ◽

Tsutomu Sekizaki ◽

Yuichi Yokomizo

Keyword(s):

Virulent Strain ◽

Broth Culture ◽

Erysipelothrix Rhusiopathiae ◽

Specific Detection ◽

First Line ◽

Enrichment Broth ◽

Dna Fragment ◽

Conventional Methods ◽

Swine Erysipelas ◽

Highly Virulent

We have previously described the creation by Tn916mutagenesis of avirulent transposition mutants from a highly virulent strain of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas. In this study, we cloned a 2.2-kb DNA fragment which flanked the Tn916 insertion in an avirulent mutant (strain 33H6) and evaluated the possibility that this region could be used for the specific detection of E. rhusiopathiae. According to the sequences of this region, oligonucleotide primers were designed to amplify a 937-bp fragment of the E. rhusiopathiae chromosome by PCR. The specificity of the PCR was investigated by analyzing 64 strains of Erysipelothrixspecies and 27 strains of other genera different fromErysipelothrix. A 937-bp DNA fragment could be amplified from all E. rhusiopathiae strains tested, and no amplification was observed by using DNAs from the other species tested. To make a rapid and definite diagnosis of swine erysipelas in slaughterhouses, we developed an enrichment broth cultivation-PCR combination assay, which used a commercially available DNA extraction kit, to identify E. rhusiopathiae in the specimens from swine with arthritis. After samples were enriched in selective broth culture, detection of E. rhusiopathiae was tested by either conventional methods or the PCR. Of 102 samples tested, 15 samples were positive by conventional methods and 12 of the 15 samples were positive by the PCR. The detection limit of the PCR was 103 CFU per reaction mixture for the PCR-positive samples. These results indicate that this PCR technique could be used as a first-line screening technique for the specific detection of E. rhusiopathiae in specimens.

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