multiplex pcrs
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2021 ◽  
Vol 255 ◽  
pp. 109021
Author(s):  
Oliver W. Stringer ◽  
Janine T. Bossé ◽  
Sonia Lacouture ◽  
Marcelo Gottschalk ◽  
László Fodor ◽  
...  

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Rebecca M. Mitchell ◽  
Zhiyong Zhou ◽  
Mili Sheth ◽  
Sheila Sergent ◽  
Michael Frace ◽  
...  

Abstract Background Simultaneous infection with multiple malaria parasite strains is common in high transmission areas. Quantifying the number of strains per host, or the multiplicity of infection (MOI), provides additional parasite indices for assessing transmission levels but it is challenging to measure accurately with current tools. This paper presents new laboratory and analytical methods for estimating the MOI of Plasmodium falciparum. Methods Based on 24 single nucleotide polymorphisms (SNPs) previously identified as stable, unlinked targets across 12 of the 14 chromosomes within P. falciparum genome, three multiplex PCRs of short target regions and subsequent next generation sequencing (NGS) of the amplicons were developed. A bioinformatics pipeline including B4Screening pathway removed spurious amplicons to ensure consistent frequency calls at each SNP location, compiled amplicons by SNP site diversity, and performed algorithmic haplotype and strain reconstruction. The pipeline was validated by 108 samples generated from cultured-laboratory strain mixtures in different proportions and concentrations, with and without pre-amplification, and using whole blood and dried blood spots (DBS). The pipeline was applied to 273 smear-positive samples from surveys conducted in western Kenya, then providing results into StrainRecon Thresholding for Infection Multiplicity (STIM), a novel MOI estimator. Results The 24 barcode SNPs were successfully identified uniformly across the 12 chromosomes of P. falciparum in a sample using the pipeline. Pre-amplification and parasite concentration, while non-linearly associated with SNP read depth, did not influence the SNP frequency calls. Based on consistent SNP frequency calls at targeted locations, the algorithmic strain reconstruction for each laboratory-mixed sample had 98.5% accuracy in dominant strains. STIM detected up to 5 strains in field samples from western Kenya and showed declining MOI over time (q < 0.02), from 4.32 strains per infected person in 1996 to 4.01, 3.56 and 3.35 in 2001, 2007 and 2012, and a reduction in the proportion of samples with 5 strains from 57% in 1996 to 18% in 2012. Conclusion The combined approach of new multiplex PCRs and NGS, the unique bioinformatics pipeline and STIM could identify 24 barcode SNPs of P. falciparum correctly and consistently. The methodology could be applied to field samples to reliably measure temporal changes in MOI.


Author(s):  
Mónica Cerezales ◽  
Lea Biniossek ◽  
Stefanie Gerson ◽  
Kyriaki Xanthopoulou ◽  
Julia Wille ◽  
...  

Introduction. Gram-negative bacteria are a common source of infection both in hospitals and in the community, and antimicrobial resistance is frequent among them, making antibiotic therapy difficult, especially when these isolates carry carbapenem resistance determinants. Hypothesis/Gap Statement. A simple method to detect all the commonly found carbapenemases in Germany was not available. Aim. The aim of this study was to develop a multiplex PCR for the rapid and reliable identification of the most prevalent carbapenemase-encoding genes in Gram-negative bacteria in Germany. Methodology. Data from the German Gram-negative reference laboratory revealed the most prevalent carbapenemase groups in Germany were (in order of prevalence): bla VIM, bla OXA-48, bla OXA-23, bla KPC, bla NDM, bla OXA-40, bla OXA-58, bla IMP, bla GIM, bla GES, ISAba1-bla OXA-51, bla IMI, bla FIM and bla DIM. We developed and tested two multiplex PCRs against 83 carbapenem-resistant Gram-negative clinical isolates. Primers were designed for each carbapenemase group within conserved regions of the encoding genes obtained from publicly available databases. Multiplex-1 included the carbapenemase groups bla VIM, bla OXA-48, bla OXA-23, bla KPC, bla NDM and bla OXA-40, while multiplex-2 included bla OXA-58, bla IMP, bla GIM, bla GES, ISAba1-bla OXA-51 and bla IMI. Results. In the initial evaluation, all but one of the carbapenemases encoded by 75 carbapenemase-positive isolates were detected using the two multiplex PCRs, while no false-positive results were obtained from the remaining eight isolates. After evaluation, we tested 546 carbapenem-resistant isolates using the multiplex PCRs, and all carbapenemases were detected. Conclusion. A rapid and reliable method was developed for detection and differentiation of 12 of the most prevalent carbapenemase groups found in Germany. This method allows for the rapid testing of clinical isolates prior to species identification and does not require prior phenotypical characterization, constituting a rapid and valuable tool in the management of infections in hospitals.


Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2830-2830
Author(s):  
Michaela Kotrova ◽  
Henrik Knecht ◽  
Dietrich Herrmann ◽  
Martin Schwarz ◽  
Karin Olsen ◽  
...  

Abstract Introduction The current gold standard molecular method for minimal residual disease (MRD) assessment is RQ-PCR for detection of leukemia-associated immunoglobulin (IG) and T cell receptor (TR) rearrangements, which must be identified beforehand in the diagnostic material. Conventional screening of IG/TR rearrangements in the diagnostic sample consists of several multiplex PCRs followed by Sanger sequencing. Recently, the EuroClonality-NGS Consortium (www.euroclonalityngs.org, coordinated by AW Langerak) developed a set of assays for next-generation sequencing-based marker identification in lymphoid malignancies (Brüggemann, Haematologica, 2017). We have been using this approach in routine diagnostics since 2016 and herein we provide an overview of markers detected in acute lymphoblastic leukemia (ALL) patients. Material & Methods Between 02/2017 and 06/2018, 471 ALL samples were screened employing the EuroClonality-NGS assay. The assay consists of 5-8 multiplex PCRs, depending on the type of malignancy [IGH-VJ, IGH-DJ, TRB-VJ, TRB-DJ, TRG, TRD, and IGK-VJ-Kde (and intron-Kde) tubes] followed by a 2nd-step PCR in which barcodes and sequencing adaptors are introduced. The resulting libraries were sequenced on the Illumina MiSeq, producing 2×250bp reads, aiming to reach at least 3000 reads per sequencing library. Rearrangements with an abundance ≥ 5% were considered to be leukemia-associated. Out of the 471 investigated samples, 453 were diagnostic (dx) and 18 were relapse (rel) samples. If available, bone marrow samples (BM) were investigated (n=370), otherwise peripheral blood (PB) samples were used (n=101). For B-ALL patients (n=344) all PCRs were performed, whereas for T-ALL (n=127) the IGH-VJ and both IGK tubes were omitted. Results At least one leukemia-associated marker was detected in 244/253 (96%) B-ALL dx BM samples (Fig. 1A), 237 (94%) of patients carried two or more rearrangements. On average we detected 5.1 markers per patient (range 0-10). The majority of patients carried at least one complete IGH (168; 66%), TRG (155; 61%), TRD (154; 61%), or IGK (142; 56%) rearrangement (Fig. 1B). In dx BM T-ALL samples at least one leukemic marker was detected in 88/100 (88%) cases, and in 80 cases (80%) two or more were detected (Fig. 1C). On average 3.6 markers per patient were detected (range 0-10). The majority of patients carried TRG (67; 67%), TRD (65; 65%), or complete TRB (52; 52%) rearrangements (Fig. 1D). In dx PB samples at least 1 marker was detected in 74/75 (98%) B-ALL (on average 5.2 markers per patient), and 23/25 (92%) T-ALL samples (on average 3.7 markers per patient). Out of 18 rel samples (17 BM, 1 PB; 16 B-ALL, 2 T-ALL), 14 (78%) carried at least 1 rearrangement and 13 (72%) at least two rearrangements. On average 3.7 markers per patient were detected. Conclusions The EuroClonality-NGS assays detected at least 1 leukemic marker in 443/471 (94%) patients in our cohort. The number of patients with no marker was higher in the T-ALL cohort (15/127; 12%) compared to the B-ALL cohort (13/344; 4%). Only 3/100 (3%) dx PB samples had no marker detected, which is surprising considering the usually lower leukemic infiltration of PB in B-ALL, but can be attributed to the higher sensitivity of NGS. Besides higher sensitivity, NGS-based marker identification brings along other benefits: the turn-around time is shorter and it is possible to detect multiple markers per tube without laborious cloning, or splitting the multiplex PCRs into singleplex. Overall, EuroClonality-NGS assays have been shown to be robust, sensitive, and routinely applicable in a clinical diagnostic setting. Disclosures Goekbuget: Kite / Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Other: Travel support, Research Funding; Pfizer: Consultancy, Other: Travel support, Research Funding; Amgen: Consultancy, Other: Travel support, Research Funding. Brüggemann:PRMA: Consultancy; Pfizer: Speakers Bureau; Roche: Speakers Bureau; Regeneron: Research Funding; Affimed: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Incyte: Consultancy.


2017 ◽  
pp. 190-196
Author(s):  
Thi Anh Ngoc Le ◽  
Nu Xuan Thanh Le ◽  
Thi Nam Lien Nguyen ◽  
Viet Quynh Tram Ngo ◽  
Santon Antonella ◽  
...  

Introduction: Acinetobacter baumannii resistant to carbapenem is the most common cause of nosocomial infection, they are recognized as one of the most difficult to control and treat resistant agents. A. baumannii is the most important member of A. calcoaceticus- A. baumannii complex (ACB complex), which are closely related genetically then biochemical test systems are not able to identify unambiguously genomic species. Objective: To identify species of Acinetobacter sp. strains of ACB complex and sequence types of A. baumannii strains. Methods: Cross- sectional study. The subjects were 90 strains of ACB complex isolated and identified by biochemical methods at Hue University Hospital (UH) and Hue Central Hospital (CH). Performing the multiplex PCRs on the target gene gyrB and sequencing rpoB gene to identify species. Determining of antibiotic resistance by Kirby-Bauer method. Multi Locus Sequence Type (MLST) method was performed for 24 strains of A. baumannii to identify sequence types. Results: Acinetobacter sp. were identified as A.bau-mannii (85.6%), A. nosocomialis (11.11%) and A. pittii (3.33%). All strains of ACB complex were sensitive to colistin and resistant to cefotaxime, amikacin. More than 90% of A.baumannii strains were resistant to carbapenem. Eleven sequence types (STs) of A.baumannii strains were identified; two of them were news (ST1463 and ST1464) and seven STs belonged to the Clonal Complex 92 (CC92), the most globally distributed clones; the ST795, ST797 and ST1463 are circulating in both of hospitals. Conclusion: Combining multiplex PCRs on the target gene gyrB and sequencing rpoB gene have shown to be adequate for Acinetobacter sp.identification. MLST identified 11 sequence types from 24 A.baumannii isolates at two hospital in Hue. Key words: ACB complex, Acinetobacter baumannii, MLST


2015 ◽  
Vol 11 (2) ◽  
Author(s):  
Belkacem Zarouri ◽  
Alba María Vargas ◽  
Laura Gaforio ◽  
María Aller ◽  
María Teresa de Andrés ◽  
...  

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