Single-copy detection of somatic variants from solid and liquid biopsy

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

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Real-time evaluation of an optimized real-time PCR assay versus Brilliance chromogenic MRSA agar for the detection of meticillin-resistant Staphylococcus aureus from clinical specimens

Journal of Medical Microbiology ◽

10.1099/jmm.0.025288-0 ◽

2011 ◽

Vol 60 (3) ◽

pp. 323-328 ◽

Cited By ~ 12

Author(s):

J. Danial ◽

M. Noel ◽

K. E. Templeton ◽

F. Cameron ◽

F. Mathewson ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Real Time ◽

Real Time Pcr ◽

Test Performance ◽

High Sensitivity ◽

Turnaround Time ◽

Homology Search ◽

Pcr Assay ◽

Indirect Measure ◽

The Mean

A total of 1204 meticillin-resistant Staphylococcus aureus (MRSA) screens (3340 individual swabs) were tested to evaluate a staphylococcal cassette chromosome mec (SCCmec) real-time PCR. In total, 148 (12.3 %) of the screens were MRSA-positive, where 146 (12.1 %) were MRSA-positive by the SCCmec real-time PCR assay. In contrast, 128 (10.6 %) screens were MRSA-positive by culture. One hundred and twenty-six (10.5 %) of the screens were positive by both culture and PCR. Twenty of the 1204 screens (1.66 %) were negative by culture but positive by PCR; these samples were sequenced. In 14 of the cases, a homology search confirmed the sequence as SCCmec, indicating that these samples could be considered true positives. Two of the 1204 (0.2 %) screens were positive by culture and negative by PCR. The mean turnaround time (TAT) for PCR-negative swabs was 6 h 12 min and for PCR-positive swabs was 6 h 48 min. In comparison, for culture-negative swabs the mean TAT was 29 h 30 min and for culture-positive swabs was 69 h. The cost per swab for routine culture was £0.41 (€0.48) and that of the real-time PCR assay was £2.35 (€2.75). This optimized, in-house, inexpensive, real-time PCR test maintained a very high sensitivity and specificity when evaluated under real-time laboratory conditions. The TAT of this real-time PCR assay was substantially lower than that of chromogenic culture. It was also maintained throughout the entire process, which can be taken as an indirect measure of test performance. This study showed that implementation of a molecular test can be achieved with limited resources in a standard microbiology laboratory.

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Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species

Microbiology Insights ◽

10.4137/mbi.s38517 ◽

2016 ◽

Vol 9 ◽

pp. MBI.S38517 ◽

Cited By ~ 15

Author(s):

Jing Zhang ◽

Guo-Chiuan Hung ◽

Kenjiro Nagamine ◽

Bingjie Li ◽

Shien Tsai ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Candida Species ◽

Rapid Detection ◽

Corneal Transplantation ◽

High Sensitivity ◽

Turnaround Time ◽

Pcr Assays ◽

Primer Sets ◽

Pcr Primer

Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan- Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan- Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

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Detection of soybean by real-time PCR in the samples subjected to deep technological processing

Theory and practice of meat processing ◽

10.21323/2414-438x-2019-4-4-23-27 ◽

2019 ◽

Vol 4 (4) ◽

pp. 23-27

Author(s):

K. A. Kurbakov ◽

E. A. Konorov ◽

V. N. Zhulinkova ◽

M. Yu. Minaev

Keyword(s):

Glycine Max ◽

Real Time ◽

Real Time Pcr ◽

High Sensitivity ◽

Food Product ◽

Single Copy ◽

Probe System ◽

Canned Foods ◽

Technological Processing ◽

The Difference

During deep technological processing, DNA of food product components (specifically, in canned foods) is subjected to strong degradation, which makes the PCR-based food components identification more difficult. In this work, a primer-probe system is proposed, which was selected for the multi-copy region of long terminal repeat (LTR) of soybean (Glycine max). We confirmed its high sensitivity and specificity for soybean detection by real-time PCR. Using the selected system, we successfully analyzed the samples of meat-and-plant canned foods and other food products subjected to deep technological processing — tofu, preserved tofu, soy sauces, confectionary products containing soy lecithin. To compare with these samples, real-time PCR was carried out using the primer-probe system selected for the single-copy le1 gene. In terms of sensitivity, the use of the primer-probe system specific to the single-copy region was significantly inferior to the primer-probe system specific to the LTR region. The difference in the rate of degradation of these genomic DNA regions of Glycine max was found.

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Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates

Brazilian Journal of Biology ◽

10.1590/1519-6984.229893 ◽

2020 ◽

Author(s):

F. Alexandrino ◽

J. S. Malgarin ◽

M. A. Krieger ◽

L. G. Morello

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

High Sensitivity ◽

Turnaround Time ◽

Storage Conditions ◽

Rrna Gene ◽

Platelet Concentrates ◽

Microbiological Methods ◽

Bacterial Screening

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.

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COLD-PCR: a new platform for highly improved mutation detection in cancer and genetic testing

Biochemical Society Transactions ◽

10.1042/bst0370427 ◽

2009 ◽

Vol 37 (2) ◽

pp. 427-432 ◽

Cited By ~ 52

Author(s):

Jin Li ◽

G. Mike Makrigiorgos

Keyword(s):

Genetic Testing ◽

Real Time ◽

Single Molecule ◽

Real Time Pcr ◽

Clinical Decision Making ◽

Mutation Detection ◽

Detection Sensitivity ◽

Detection Methods ◽

Wild Type ◽

Variant Alleles

PCR is widely employed as the initial DNA amplification step for genetic testing and cancer biomarker detection. However, a key limitation of PCR-based methods, including real-time PCR, is the inability to selectively amplify low levels of variant alleles in a wild-type allele background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact on clinical decision-making and outcome or that can serve as cancer biomarkers. We developed COLD-PCR (co-amplification at lower denaturation temperature-PCR) [Li, Wang, Mamon, Kulke, Berbeco and Makrigiorgos (2008) Nat. Med. 14, 579–584], a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. Consequently, COLD-PCR amplification from genomic DNA yields PCR products containing high-prevalence variant alleles that can be detected. Since PCR constitutes a ubiquitous initial step for almost all genetic analysis, COLD-PCR provides a general platform to improve the sensitivity of essentially all DNA-variation detection technologies including Sanger sequencing, pyrosequencing, single molecule sequencing, mutation scanning, mutation genotyping or methylation assays. COLD-PCR combined with real-time PCR provides a new approach to boost the capabilities of existing real-time mutation detection methods. We replaced regular PCR with COLD-PCR before sequencing or real-time mutation detection assays to improve mutation detection-sensitivity by up to 100-fold and identified novel p53/Kras/EGFR (epidermal growth factor receptor) mutations in heterogeneous cancer samples that were missed by all existing methods. For clinically relevant micro-deletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR is expected to have diverse applications in the fields of biomarker identification and tracing, genomic instability, infectious diseases, DNA methylation testing and prenatal identification of fetal alleles in maternal blood.

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Laboratory predictors of death from coronavirus disease 2019 (COVID-19) in the area of Valcamonica, Italy

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2020-0459 ◽

2020 ◽

Vol 58 (7) ◽

pp. 1100-1105 ◽

Cited By ~ 16

Author(s):

Graziella Bonetti ◽

Filippo Manelli ◽

Andrea Patroni ◽

Alessandra Bettinardi ◽

Gianluca Borrelli ◽

...

Keyword(s):

Hospital Stay ◽

Clinical Decision Making ◽

Troponin I ◽

Laboratory Data ◽

High Sensitivity ◽

Clinical Decision ◽

International Normalized Ratio ◽

Serum Glucose ◽

Hospital Death ◽

Laboratory Findings

AbstractBackgroundComprehensive information has been published on laboratory tests which may predict worse outcome in Asian populations with coronavirus disease 2019 (COVID-19). The aim of this study is to describe laboratory findings in a group of Italian COVID-19 patients in the area of Valcamonica, and correlate abnormalities with disease severity.MethodsThe final study population consisted of 144 patients diagnosed with COVID-19 (70 who died during hospital stay and 74 who survived and could be discharged) between March 1 and 30, 2020, in Valcamonica Hospital. Demographical, clinical and laboratory data were collected upon hospital admission and were then correlated with outcome (i.e. in-hospital death vs. discharge).ResultsCompared to patients who could be finally discharged, those who died during hospital stay displayed significantly higher values of serum glucose, aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH), urea, creatinine, high-sensitivity cardiac troponin I (hscTnI), prothrombin time/international normalized ratio (PT/INR), activated partial thromboplastin time (APTT), D-dimer, C reactive protein (CRP), ferritin and leukocytes (especially neutrophils), whilst values of albumin, hemoglobin and lymphocytes were significantly decreased. In multiple regression analysis, LDH, CRP, neutrophils, lymphocytes, albumin, APTT and age remained significant predictors of in-hospital death. A regression model incorporating these variables explained 80% of overall variance of in-hospital death.ConclusionsThe most important laboratory abnormalities described here in a subset of European COVID-19 patients residing in Valcamonica are highly predictive of in-hospital death and may be useful for guiding risk assessment and clinical decision-making.

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Real-Time PCR for Molecular Detection of Zoonotic and Non-Zoonotic Giardia spp. in Wild Rodents

Microorganisms ◽

10.3390/microorganisms9081610 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1610

Author(s):

Christian Klotz ◽

Elke Radam ◽

Sebastian Rausch ◽

Petra Gosten-Heinrich ◽

Toni Aebischer

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Gastrointestinal Disease ◽

Single Copy ◽

Marker Genes ◽

Small Rodent ◽

Analytical Sensitivity ◽

Single Copy Gene ◽

Wild Rodents ◽

Copy Gene

Giardiasis in humans is a gastrointestinal disease transmitted by the potentially zoonotic Giardia duodenalis genotypes (assemblages) A and B. Small wild rodents such as mice and voles are discussed as potential reservoirs for G. duodenalis but are predominantly populated by the two rodent species Giardia microti and Giardia muris. Currently, the detection of zoonotic and non-zoonotic Giardia species and genotypes in these animals relies on cumbersome PCR and sequencing approaches of genetic marker genes. This hampers the risk assessment of potential zoonotic Giardia transmissions by these animals. Here, we provide a workflow based on newly developed real-time PCR schemes targeting the small ribosomal RNA multi-copy gene locus to distinguish G. muris, G. microti and G. duodenalis infections. For the identification of potentially zoonotic G. duodenalis assemblage types A and B, an established protocol targeting the single-copy gene 4E1-HP was used. The assays were specific for the distinct Giardia species or genotypes and revealed an analytical sensitivity of approximately one or below genome equivalent for the multi-copy gene and of about 10 genome equivalents for the single-copy gene. Retesting a biobank of small rodent samples confirmed the specificity. It further identified the underlying Giardia species in four out of 11 samples that could not be typed before by PCR and sequencing. The newly developed workflow has the potential to facilitate the detection of potentially zoonotic and non-zoonotic Giardia species in wild rodents.

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Integrated Extreme Real-Time PCR and High-Speed Melting Analysis in 52 to 87 Seconds

Clinical Chemistry ◽

10.1373/clinchem.2018.296608 ◽

2019 ◽

Vol 65 (2) ◽

pp. 263-271 ◽

Cited By ~ 8

Author(s):

Joseph T Myrick ◽

Robert J Pryor ◽

Robert A Palais ◽

Sean J Ison ◽

Lindsay Sanford ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

High Speed ◽

Dna Analysis ◽

Point Of Care ◽

Turnaround Time ◽

Single Nucleotide Variants ◽

Cycle Times ◽

Pcr Products ◽

Melting Analysis

Abstract BACKGROUND Extreme PCR in <30 s and high-speed melting of PCR products in <5 s are recent advances in the turnaround time of DNA analysis. Previously, these steps had been performed on different specialized instruments. Integration of both extreme PCR and high-speed melting with real-time fluorescence monitoring for detection and genotyping is presented here. METHODS A microfluidic platform was enhanced for speed using cycle times as fast as 1.05 s between 66.4 °C and 93.7 °C, with end point melting rates of 8 °C/s. Primer and polymerase concentrations were increased to allow short cycle times. Synthetic sequences were used to amplify fragments of hepatitis B virus (70 bp) and Clostridium difficile (83 bp) by real-time PCR and high-speed melting on the same instrument. A blinded genotyping study of 30 human genomic samples at F2 c.*97, F5 c.1601, MTHFR c.665, and MTHFR c.1286 was also performed. RESULTS Standard rapid-cycle PCR chemistry did not produce any product when total cycling times were reduced to <1 min. However, efficient amplification was possible with increased primer (5 μmol/L) and polymerase (0.45 U/μL) concentrations. Infectious targets were amplified and identified in 52 to 71 s. Real-time PCR and genotyping of single-nucleotide variants from human DNA was achieved in 75 to 87 s and was 100% concordant to known genotypes. CONCLUSIONS Extreme PCR with high-speed melting can be performed in about 1 min. The integration of extreme PCR and high-speed melting shows that future molecular assays at the point of care for identification, quantification, and variant typing are feasible.

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