Polymerase Chain Reaction Platform Research Articles

Sensitive detection of multiple blood biomarkers via immunomagnetic exosomal PCR for the diagnosis of Alzheimer's disease.

Blood exosomes are emerging as potential biomarkers for diagnosing brain diseases such as Alzheimer's disease (AD). There is currently a lack of an ultrasensitive technology for identifying core AD biomarkers in blood exosomes to optimize the utility of biomarkers in clinical practice. Here, an immunomagnetic exosomal polymerase chain reaction (iMEP) platform was developed using DNA-conjugated antibodies for the rapid detection of amyloid-β (Aβ1-40 and Aβ1-42) and phosphorylated tau (p-tau396,404 and p-tau181) in clinical blood exosomes. The toehold shift-mediated DNA affinity pulldown eliminates the high detection background, which allows the detection of biomarkers at concentrations down to 10 femtograms per milliliter. With the iMEP assay, exosomal Aβ1-42 was more accurate in differentiating patients with AD from healthy individuals compared with exosomal p-tau181 and p-tau396,404, with a sensitivity of 95.0% and a specificity of 95.0%. The iMEP technique is also adept at quantifying the levels of different exosomal biomarkers associated with disease pathogenesis.

Abstract 3780: From diagnostic labs to rapid near-patient testing: Bridging across RT-qPCR platforms of a clinicopathological and gene expression model for cutaneous melanoma patients

Abstract Background: Gene expression signatures are becoming increasingly common in clinical settings, for diagnosis, risk stratification, and treatment response prediction. However, deployment in clinical practice often entails a molecular platform different from the one used for development. Transporting molecular signatures across platforms (“bridging”) cannot be achieved directly, due to differences in scales and distributions in gene expression and requires bespoke methods. Here, we focused on the bridging of a clinicopathological and gene expression model (CP-GEP) across two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) platforms: a single-plex SYBR Green qPCR, and a multiplex TaqMan probe-based qPCR (Idylla TM platform). CP-GEP is a regression model, combining eight genes (plus two housekeeping genes) and two clinicopathologic variables (age and Breslow thickness), able to stratify patients with melanoma into two groups based on their risk of sentinel lymph node metastasis. Methods: For the bridging, we selected 150 samples of primary melanoma patients who underwent a sentinel lymph node biopsy from a cohort of stage I-III melanoma patients. We measured the expression of the eight genes on both qPCR platforms and quantified them as ∆Ct. Within a bootstrapping scheme (500 repeats), for each gene, we learned a mapping (“bridging function”) between ∆Ct spaces, using all eight genes as regressors. Exploiting the linearity of per-gene bridging functions, we reparametrized the CP-GEP model so that we could directly use it in the target platform. We assessed the performance of the bridging, in terms of Percent Agreement (PA), defined as the percentage of the concordant binary outputs between the original and the bridged models; both in the bootstrapped samples and in 15 samples from an independent cohort. Results: In the bootstrapped samples, we obtained a PA of 100%, in the intervals for which we had pre-defined acceptance criteria, based on the distance from the cut point; and we had a mean PA of 89.2% [95% CI, 85.23%, 94.6%] when we considered all samples. In the external validation, we achieved a PA of 100%; all performance metrics were identical. The continuous scores of the original and bridged models were in excellent agreement, with an R2 equal to 0.95 both in the bridging and external validation cohorts. Conclusions: We showed that a model, combining gene expression and clinicopathologic variables, originally developed on a single-plex SYBR Green RT-qPCR, often used in diagnostic labs, could be robustly reparametrized to be compatible with a multiplex TaqMan probe-based qPCR (Idylla TM platform), more suitable for point-of-care-diagnostics. The ability to transfer models across different molecular platforms can greatly accelerate the translation of fundamental research to clinical practice. Citation Format: Lara Pozza, Sena Zumrutcu, Lizanne Bosman, Ashley van der Spek, Melanie Moreau, Dennie Tempel, Jvalini Dwarkasing, Domenico Bellomo. From diagnostic labs to rapid near-patient testing: Bridging across RT-qPCR platforms of a clinicopathological and gene expression model for cutaneous melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3780.

Comparing the performance of 2 human papillomavirus assays for a new use indication: a real-world evidence-based evaluation in the United States

The US Food and Drug Administration (FDA) supports innovations to facilitate new indications for high-risk human papillomavirus (HPV) testing. This report describes the retrospective testing of stored specimens and analysis of existing data to efficiently and cost-effectively support a new indication for the Onclarity HPV assay (Becton, Dickinson and Company; BD Life Sciences Integrated Diagnostic Solutions; Sparks, MD, USA). The performance of this index test was compared to a predicate test, the cobas HPV assay (Roche Diagnostics; Indianapolis, IN, USA). Both HPV assays are based on real-time polymerase chain reaction (PCR) platforms that detect the presence of 14 high-risk HPV genotypes. The predicate assay reports HPV16 and 18 as individual results and the other 12 HPV genotypes as one pooled result. The index assay reports nine, independent results (HPV 16, 18, 31, 33/58, 35/39/68, 45, 51, 52, 56/59/66). The index and predicate assays are both FDA-approved for cervical cancer screening but at the time that this study was initiated, the index HPV assay was not approved for use with cervical specimens collected in PreservCyt (Hologic, Inc.; San Diego, CA, USA) liquid-based cytology (LBC) media. The performance of the index HPV assay was compared to the predicate HPV assay for detection of cervical intraepithelial neoplasia grades 2 or 3 or worse (≥CIN2 or ≥CIN3) using PreservCyt LBC specimens collected from women ages 21-65 years. The ability of the index test's extended genotyping to stratify ≥CIN2 or ≥CIN3 risks, using these specimens, was also evaluated. The New Mexico HPV Pap Registry was used to select an age- and cytology-stratified random sample of 19,879 women undergoing opportunistic cervical screening and follow-up in routine clinical practice across New Mexico. A random subset of PreservCyt specimens from 4,820 of these women was selected for paired testing by the index and predicate HPV assays within age and cytology strata and included women with or without cervical biopsy follow-up. Point estimate differences and ratios were calculated for cervical disease detection and positivity rates, respectively, with 95% confidence intervals (95% CI) to determine statistical significance. The cumulative risk of ≥CIN2 or ≥CIN3, with up to five-years follow-up, was estimated for the index assay using Kaplan-Meier methods. Five-year cumulative ≥CIN3 detection was 5.6% and 4.6% for the index and predicate assay, respectively (difference=1.0% [.5% to 1.5%]). The ≥CIN3 positivity rate within <1-year for the index and predicate assay was 95.3% and 94.5%, respectively (ratio=1.01 [.98 to 1.06]). The ≥CIN3 cumulative positivity rates for the index and predicate assays were also similar at five-years. Among cases of ≥CIN3, positive agreement between the index and predicate assay for HPV16 and HPV18, was 100% (95%CI=95.0% to 100%), and 90.9% (95%CI=62.3% to 98.4%), respectively. HPV16 carried the highest ≥CIN2/3 risk, followed by HPV18/31/33/58/52/45, and then HPV35/56/59/51/56/59/66. The index and predicate HPV assays demonstrated equivalent performance, and extended HPV genotyping, using the index assay, provided effective ≥CIN2 and ≥CIN3 risk stratification, supporting a new indication for use of the index assay with PreservCyt.

Discrimination of SARS-CoV-2 omicron variant and its lineages by rapid detection of immune-escape mutations in spike protein RBD using asymmetric PCR-based melting curve analysis

BackgroundThe SARS-CoV-2 Omicron strain has multiple immune-escape mutations in the spike protein receptor-binding domain (RBD). Rapid detection of these mutations to identify Omicron and its lineages is essential for guiding public health strategies and patient treatments. We developed a two-tube, four-color assay employing asymmetric polymerase chain reaction (PCR)-based melting curve analysis to detect Omicron mutations and discriminate the BA.1, BA.2, BA.4/5, and BA.2.75 lineages.MethodsThe presented technique involves combinatory analysis of the detection of six fluorescent probes targeting the immune-escape mutations L452R, N460K, E484A, F486V, Q493R, Q498R, and Y505H within one amplicon in the spike RBD and probes targeting the ORF1ab and N genes. After protocol optimization, the analytical performance of the technique was evaluated using plasmid templates. Sensitivity was assessed based on the limit of detection (LOD), and reliability was assessed by calculating the intra- and inter-run precision of melting temperatures (Tms). Specificity was assessed using pseudotyped lentivirus of common human respiratory pathogens and human genomic DNA. The assay was used to analyze 40 SARS-CoV-2–positive clinical samples (including 36 BA.2 and 4 BA.4/5 samples) and pseudotyped lentiviruses of wild-type and BA.1 viral RNA control materials, as well as 20 SARS-CoV-2–negative clinical samples, and its accuracy was evaluated by comparing the results with those of sequencing.ResultsAll genotypes were sensitively identified using the developed method with a LOD of 39.1 copies per reaction. The intra- and inter-run coefficients of variation for the Tms were ≤ 0.69% and ≤ 0.84%, with standard deviations ≤ 0.38 °C and ≤ 0.41 °C, respectively. Validation of the assay using known SARS-CoV-2–positive samples demonstrated its ability to correctly identify the targeted mutations and preliminarily characterize the Omicron lineages.ConclusionThe developed assay can provide accurate, reliable, rapid, simple and low-cost detection of the immune-escape mutations located in the spike RBD to detect the Omicron variant and discriminate its lineages, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.

Viral load: We need a new look at an old problem?

The concept of viral load was introduced in the 1980s to measure the amount of viral genetic material in a person's blood, primarily for human immunodeficiency virus (HIV). It has since become crucial for monitoring HIV infection progression and assessing the efficacy of antiretroviral therapy. However, during the coronavirus disease 2019 pandemic, the term "viral load" became widely popularized, not only for the scientific community but for the general population. Viral load plays a critical role in both clinical patient management and research, providing valuable insights for antiviral treatment strategies, vaccination efforts, and epidemiological control measures. As measuring viral load is so important, why don't researchers discuss the best way to do it? Is it simply acceptable to use raw Ct values? Relying solely on Ct values for viral load estimation can be problematic due to several reasons. First, Ct values can vary between different quantitative polymerase chain reaction assays, platforms, and laboratories, making it difficult to compare data across studies. Second, Ct values do not directly measure the quantity of viral particles in a sample and they can be influenced by various factors such as initial viral load, sample quality, and assay sensitivity. Moreover, variations in viral RNA extraction and reverse-transcription steps can further impact the accuracy of viral load estimation, emphasizing the need for careful interpretation of Ct values in viral load assessment. Interestingly, we did not observe scientific articles addressing different strategies to quantify viral load. The absence of standardized and validated methods impedes the implementation of viral load monitoring in clinical management. The variability in cell quantities within samples and the variation in viral particle numbers within infected cells further challenge accurate viral load measurement and interpretation. To advance the field and improve patient outcomes, there is an urgent need for the development and validation of tailored, standardized methods for precise viral load quantification.

Data usefulness in determining cause of stillbirth in South Asia.

To evaluate the usefulness of data to determine cause of stillbirth in India and Pakistan. Prospective, observational study. Study hospitals in India and Pakistan. 200 fetal deaths with placental evaluation and minimally invasive tissue sampling (MITS) of internal organs and polymerase chain reaction (PCR) test for 75 pathogens. Data defined as useful to determine stillbirth causes. Placental pathology was the most useful to determine cause of stillbirth. Comparing placental and fetal weight with standard weights was useful in 44.5% and 48.5%, respectively. Lung histology was useful in 42.5%. Most of the other findings of internal organ histology were only occasionally useful. Signs of abruption, by maternal history or placental evaluation, were always deemed useful. Placenta, brain and cord blood PCR were also useful, but less often than histology. Based on this analysis, maternal clinical history, placental histology and fetal examination were most informative. Comparing the placental and fetal weights with recognised standards was useful in nearly half the cases. Fetal tissue histology and PCR were also informative. Of all the potential tests of MITS-obtained specimens, we would first recommend histological evaluation of the lungs, and using a multiplex PCR platform would determine pathogens in blood and brain/CSF. We recognise that this approach will not identify some causes, including some genetic and internal organ anomalies, but will confirm most common causes of stillbirth and most of the preventable causes of stillbirth in low- and middle-income countries.

Comparison of digital PCR platforms using the molecular marker.

Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.

Best practices in epidermal growth factor receptor T790M testing for advanced non-small-cell lung cancer in Hong Kong.

The T790M mutation in the epidermal growth factor receptor gene causes most acquired resistance to firstor second-line epidermal growth factor receptor-tyrosine kinase inhibitors in advanced non-small-cell lung cancer. The results of T790M testing can guide subsequent treatment. Despite the availability of guidelines from international organisations, T790M testing practices in Hong Kong must be streamlined and adapted to the Hospital Authority setting. To address this issue, a panel of experts in oncology and pathology met for discussion of key topics regarding T790M testing practices in Hong Kong, including the appropriate timing of testing and re-testing, as well as optimal testing methods. All panel members voted on the results of the discussion to achieve consensus. Items supported by a majority vote were adopted as consensus statements regarding current best practices for T790M testing in Hong Kong. Among the topics discussed, the panel agreed that T790M testing should be initiated upon radiological progression, including symptomatic disease progression or central nervous system-only progression. The experts also preferred initial testing with liquid biopsy, using the widely available digital polymerase chain reaction platform. This document provides the final consensus statements, as well as a testing and treatment workflow, for clinicians in Hong Kong to use as guidance in T790M testing.

Comparison of the AmpFire and MA-6000 polymerase chain reaction platforms for high-risk human papillomavirus testing in cervical precancer screening

High-risk human papillomavirus (hr-HPV) testing for primary cervical precancer screening offers an opportunity to improve screening in low-middle income countries (LMICs). This study aimed to compare the analytic performances of the AmpFire and MA-6000 platforms for hr-HPV DNA testing in three groups of women screened for hr-HPV types in Ghana: group 1 with 33 GeneXpert-archived ThinPrep/liquid-based samples subjected to both tests, group 2 with 50 AmpFire-archived dry brush samples subjected to MA-6000 testing, and group 3 involving 143 cotton swab samples simultaneously subjected to both tests without archiving. The overall agreement rates were 73 %, 92 %, and 84 %, for groups 1–3, respectively, and 84 % (95 % CI, 78.6–88.6) for the entire group. Neither AmpFire nor MA-6000 was more likely to test hr-HPV positive in all three groups and the combined group. Group 1 showed fair agreement without statistical significance (κ = 0.224, 95 % CI, −0.118 to 0.565), while group 3 showed significant moderate agreement (κ = 0.591, 95% CI, 0.442–0.741). Group 2 showed an almost perfect significant level of agreement (κ = 0.802; 95 % CI, 0.616–0.987). Thus, both platforms showed statistically significant moderate to near-perfect agreement for detecting hr-HPV in cervicovaginal samples, with variation according to archiving conditions and duration between sample collection and retesting. For LMICs using these platforms for COVID-19 testing, as the COVID-19 pandemic subsides, the platforms can become available for running other tests such as hr-HPV DNA testing for cervical precancer screening.

The power of digital PCR in fetal blood group genotyping: a review

: There is a clinical need to determine the fetal blood group status for pregnant women who present with clinically significant red cell antibodies to optimize perinatal care. Non-invasive prenatal testing (NIPT) to assess the fetal blood group status is problematic for blood groups that arise from single nucleotide variants (SNVs) because the cell-free DNA from the maternal allele is present in greater abundance than the fetal allele, resulting in competition, especially in traditional real-time quantitative polymerase chain reaction (qPCR) platforms. This review discusses the power for digital (d)PCR to overcome this limitation. The dPCR is based on first diluting and compartmentalizing individual DNA molecules into individual PCR mixes, so that the competing (e.g., maternal) molecules are separated from the target (fetal) DNA molecule prior to amplification. In comparison to qPCR platforms, dPCR is amenable to the quantitation of a positive signal from an individual DNA molecule, providing the ability to calculate the fractional abundance of the fetal sequence of interest. Comparison studies, though small in number, show that dPCR is more precise and sensitive. Clinical feasibility studies to date show that dPCR for fetal Kell antigen, Rhc, RhE and Duffy antigens have high accuracy. The key limitation of the dPCR approach is that separate assays need to be established and validated for each blood group antigen. Despite that limitation, the power of dPCR for fetal blood group genotyping provides an added diagnostic tool in the management of women with red cell antibodies.

Limitation of Interpreting a Single COVID Cycle Threshold Value

Abstract The United States reported its first case of coronavirus disease 2019 (COVID-19) in January 2020. There was limited testing capacity at the time, and the causative agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was indeed extremely infectious. Testing, therefore, has become a vital part of controlling the COVID-19 pandemic. Nucleic acid amplification tests, such as polymerase chain reaction platform, a value called cycle threshold (Ct) is reported. This number represents the number of polymerase chain reaction test cycles it takes for the sample to become positive. It is inversely proportional to the amount of viral RNA present in the sample. Therefore, a high Ct value infers a low quantity of viral RNA, and conversely, a low Ct value implies high viral RNA, which in turn correlates with infectivity. When interpreted appropriately with the right clinical context, it could be used to assess the infectiousness of an individual. In September 2021, a 73-year-old man who previously completed a primary series of Moderna vaccine (2 injections) presented to the hospital with chest pain. Cardiac catheterization showed multivessel coronary artery disease and was deemed to have unstable angina. The plan was to undergo coronary aortic bypass graft surgery. Preoperative routine testing revealed a positive COVID-19 polymerase chain reaction test with a high Ct value. During the hospital stay, it was shown that he had a decreased Ct value and thus was determined to have an acute case of asymptomatic COVID-19 infection. His surgery was delayed according to the isolation protocol per Centers for Disease Control and Prevention for safety precautions. He eventually underwent coronary aortic bypass graft after completion of isolation and had an uneventful postoperative course. It was also documented that he had serological seroconversion consistent with acute COVID-19 infection. This case illustrates that reporting Ct values is useful in a certain clinical context, but clinicians need to be familiar with the transmissibility and history of SARS-CoV-2 infection as well as understanding the interpretation of positive tests. The appropriate clinical approach and management should be assessed based on exposure history, epidemiology, physical examination, radiographic assessment, and laboratory studies.

Electroactive hydrolysis probe-based portable PCR platform for sequence-specific detection of nontyphoidal Salmonella drug resistance gene

Etaq-PCR, a portable real-time polymerase chain reaction platform, has been developed based on an electroactive hydrolysis probe design and immobilization-free electrochemical readout. The Etaq-PCR platform integrates cost effective commercial instruments and thermally stable cycling into a minimal device enabling real-time monitoring of PCR amplification. No complex electrode surface treatment or micro-fabrication is involved in the construction of the Etaq-PCR platform. Thus, one can easily construct a portable PCR platform via our design. Etaq-PCR achieved sufficient sensitivity, selectivity, and rapidness to meet the WHO ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid, and robust, equipment-free, and deliverable) for point-of-care diagnosis systems. It showed good tolerance to clinical sample matrix as it displayed 100% accuracy in a validation experiment testing a drug resistance gene of nontyphoidalSalmonella in 25 raw clinical isolated samples in comparison with the gold standard qPCR. All in all, Etaq-PCR is a portable, deployable, and cost-effective PCR platform suitable for clinical applications.

The molecular and functional characterization of ferritins in the hard tick Hyalomma rufipes

BackgroundThe protein ferritin, which plays an important role in the maintenance of iron homeostasis, is indispensable for iron detoxification, resistance to oxidative stress and innate immunity. Ticks, which are obligate blood-sucking ectoparasites, have to deal with a large amount of iron when they take a blood meal.MethodsSequence analysis was undertaken using bioinformatics. A recombinant (r) expression vector, rferritin, was constructed for a prokaryotic expression system. A quantitative polymerase chain reaction platform was used to detect the spatial and temporal expression patterns of target genes and their responses to a low temperature environment. Knockdown of the ferritin genes through RNA interference was used to analyze their effects on physiological parameters of ticks.ResultsTwo ferritin genes, HrFer1 and HrFer2, were cloned from the tick Hyalomma rufipes. Their open reading frames are 519 base pairs (bp) and 573 bp in length, and number of coding amino acids 170 and 190, respectively. The phylogenetic tree showed that HrFer1 and HrFer2 have a close evolutionary relationship with the H subunit of ferritin. In vitro experiments showed that rHrFer1 and rHrFer2 had concentration-dependent iron chelating activity. The relative expression of the two ferritin genes was higher in the ovary and midgut of H. rufipes. RNA interference results demonstrated that HrFer1 and HrFer2 expression had a significant effect on engorged body weight, number of eggs laid, and mortality of H. rufipes, and that HrFer2 also had a significant effect on feeding duration. Furthermore, the relative expression of ferritin decreased significantly in a low temperature environment, suggesting that HrFer1 and HrFer2 play a regulatory role in the cold stress response of H. rufipes.ConclusionsThe results of the present study improve our understanding of the involvement of ferritins in tick blood-feeding.Graphical

Optimized Method for the Identification of Candidate Genes and Molecular Maker Development Related to Drought Tolerance in Oil Palm (Elaeis guineensis Jacq.)

Drought is a major constraint in oil palm (Elaeis guineensis Jacq.) production. As oil palm breeding takes a long time, molecular markers of genes related to drought tolerance characteristics were developed for effective selection. Two methods of gene identification associated with drought, differential display reverse transcription polymerase chain reaction (DDRT-PCR) and pyrosequencing platform, were conducted before developing the EST-SSR marker. By DDRT-PCR, fourteen out of twenty-four primer combinations yielded the polymorphism in leaf as 77.66% and root as 96.09%, respectively. BLASTN and BLASTX revealed nucleotides from 8 out of 236 different banding similarities to genes associated with drought stress. Five out of eight genes gave a similarity with our pyrosequencing sequencing database. Furthermore, pyrosequencing analysis of two oil palm libraries, drought-tolerant, and drought sensitive, found 117 proteins associated with drought tolerance. Thirteen out of sixty EST-SSR primers could be distinguished in 119 oil palm parents in our breeding program. All of our found genes revealed an ability to develop as a molecular marker for drought tolerance. However, the function of the validated genes on drought response in oil palm must be evaluated.

Bimodal distribution pattern associated with the PCR cycle threshold (Ct) and implications in COVID-19 infections

SARS-CoV-2 is notable for its extremely high level of viral replication in respiratory epithelial cells, relative to other cell types. This may partially explain the high transmissibility and rapid global dissemination observed during the COVID-19 pandemic. Polymerase chain reaction (PCR) cycle threshold (Ct) number has been widely used as a proxy for viral load based on the inverse relationship between Ct number and amplifiable genome copies present in a sample. We examined two PCR platforms (Centers for Disease Control and Prevention 2019-nCoV Real-time RT-PCR, Integrated DNA Technologies; and TaqPath COVID-19 multi-plex combination kit, ThermoFisher Scientific) for their performance characteristics and Ct distribution patterns based on results generated from 208,947 clinical samples obtained between October 2020 and September 2021. From 14,231 positive tests, Ct values ranged from 8 to 39 and displayed a pronounced bimodal distribution. The bimodal distribution persisted when stratified by gender, age, and time period of sample collection during which different viral variants circulated. This finding may be a result of heterogeneity in disease progression or host response to infection irrespective of age, gender, or viral variants. Quantification of respiratory mucosal viral load may provide additional insight into transmission and clinical indicators helpful for infection control.

Rapid detection of the irinotecan-related UGT1A1*28 polymorphism by asymmetric PCR melting curve analysis using one fluorescent probe.

BackgroundDetermination of UGT1A1 (TA)n polymorphism prior to irinotecan therapy is necessary to avoid severe adverse drug effects. Thus, accurate and reliable genotyping methods for (TA)n polymorphism are highly desired. Here, we present a new method for polymerase chain reaction (PCR) melting curve analysis using one fluorescent probe to discriminate the UGT1A1*1 [(TA)6] and *28 [(TA)7] genotypes.MethodsAfter protocol optimization, this technique was applied for genotyping of 64 patients (including 23 with UGT1A1*1/*1, 22 with *1/*28, and 19 with *28/*28) recruited between 2016 and 2021 in China‐Japan Friendship Hospital. The accuracy of the method was evaluated by comparing the results with those of direct sequencing and fragment analysis. The intra‐ and inter‐run precision of the melting temperatures (Tms) were calculated to assess the reliability, and the limit of detection was examined to assess the sensitivity.ResultsAll genotypes were correctly identified with the new method, and its accuracy was higher than that of fragment analysis. The intra‐ and inter‐run coefficients of variation for the Tms were both ≤0.27%, with standard deviations ≤0.14°C. The limit of detection was 0.2 ng of input genomic DNA.ConclusionThe developed PCR melting curve analysis using one fluorescent probe can provide accurate, reliable, rapid, simple, and low‐cost detection of UGT1A1 (TA)n polymorphism, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.

A multiplexed electrochemical quantitative polymerase chain reaction platform for single-base mutation analysis

Detection of single-based mutation (SbM), which is of ultra-low abundance against wild-type alleles, are typically constrained by the level of multiplexing, sensitivity for single-base resolution and quantification accuracy. In this work, an electrochemical quantitative polymerase chain reaction (E-PCR) platform was developed for multiplexed and quantitative SbM analysis in limited and precious samples with single-nucleotide discrimination. A locked nucleic acid (LNA)-mediated multiplexed PCR system in a single, closed tube setup was firstly constructed to selectively amplify the SbM genes while suppressing the wild-type alleles. The amplicons were detected simultaneously through hybridization with the sequence-specific hairpin probes anchored on a reduced graphene oxide-gold nanoparticles functionalized electrode surface. With the inclusion of an LNA-mediated PCR step upstream of the electrochemical detection, we improved the limit of detection (LOD) by 2 orders of magnitude, down to an ultralow-level of 5 copies μL−1. The platform achieved an ultra-sensitive and specific detection with 0.05% against a background of 10, 000 copies of wild-type alleles. It is highly adaptive and has the potential to enable expanded multiplexed detection in parallel, thus providing a universal tool for multiplexed SbM identification.

Unique Challenges in Investigating a Cluster of COVID-19 Cases in an Inpatient Rehabilitation Unit

BackgroundThe SARS-CoV-2 virus that causes Coronavirus Infectious Disease 2019 (COVID-19) is primarily spread via droplets and aerosols when individuals are in proximity, and to a lesser degree through fomite contamination. In a six-day timeframe in January 2021 our inpatient rehabilitation unit experienced an outbreak of SARS-CoV-2 among five patients and one healthcare workerMethodsA formal outbreak investigation commenced in the setting of one hospital-acquired case of COVID-19 and evidence of transmission from a previously identified case. The investigation included contact tracing, control measures, source testing, and a descriptive epidemiology study. Testing of 26 patients and 39 employees was conducted to determine the extent of transmission. Mid-turbinate or nasopharyngeal specimens were run on QIAstat-Dx real-time reverse transcription polymerase chain reaction (RT-PCR) platform. Inpatient specimens were collected by nursing staff. Employee specimens were collected at an Urgent Care center affiliated with the health system.ResultsThree additional positive patients were discovered, one asymptomatic and two who developed symptoms. Patient activities in common included a shared dining room, shared shower facilities since many in-room showers could not accommodate a wheelchair or walker, and a physical therapy gym. One positive employee was identified through asymptomatic testing. This employee had extensive contact with the index case while the patient had cough, vomiting and diarrhea. The employee was not yet vaccinated; this was prior to implementation of an organizational vaccine requirement policy. Control measures included immediately closing shared spaces, unit-level enhanced symptom screening and employee education.ConclusionsPatients receiving physical, occupational and/or speech therapies have unique infection risks due to shared equipment of varying materials that may be difficult to clean, needing to be observed during meals for swallow safety, and removing masks during meals in the common dining room. No additional cases were identified following interventions targeted at common spaces and enhanced staff screening and education.

SAMBA: A Multicolor Digital Melting PCR Platform for Rapid Microbiome Profiling.

During the past decade, breakthroughs in sequencing technology have provided the basis for studies of the myriad ways in which microbial communities in and on the human body influence human health and disease. In almost every medical specialty, there is now a growing interest in accurate and quantitative profiling of the microbiota for use in diagnostic and therapeutic applications. However, the current next-generation sequencing approach for microbiome profiling is costly, requires laborious library preparation, and is challenging to scale up for routine diagnostics. Split, Amplify, and Melt analysis of BActeria-community (SAMBA), a novel multicolor digital melting polymerase chain reaction platform with unprecedented multiplexing capability is presented, and the capability to distinguish and quantify 16 bacteria species in mixtures is demonstrated. Subsequently, SAMBA is applied to measure the compositions of bacteria in the gut microbiome to identify microbial dysbiosis related to colorectal cancer. This rapid, low cost, and high-throughput approach will enable the implementation of microbiome diagnostics in clinical laboratories and routine medical practice.

Ultrafast DNA Amplification Using Microchannel Flow-Through PCR Device.

Polymerase chain reaction (PCR) is limited by the long reaction time for point-of-care. Currently, commercial benchtop rapid PCR requires 30–40 min, and this time is limited by the absence of rapid and stable heating and cooling platforms rather than the biochemical reaction kinetics. This study develops an ultrafast PCR (<3 min) platform using flow-through microchannel chips. An actin gene amplicon with a length of 151 base-pairs in the whole genome was used to verify the ultrafast PCR microfluidic chip. The results demonstrated that the channel of 56 μm height can provide fast heat conduction and the channel length should not be short. Under certain denaturation and annealing/extension times, a short channel design will cause the sample to drive slowly in the microchannel with insufficient pressure in the channel, causing the fluid to generate bubbles in the high-temperature zone and subsequently destabilizing the flow. The chips used in the experiment can complete 40 thermal cycles within 160 s through a design with the 56 µm channel height and with each thermal circle measuring 4 cm long. The calculation shows that the DNA extension speed is ~60 base-pairs/s, which is consistent with the theoretical speed of the Klen Taq extension used, and the detection limit can reach 67 copies. The heat transfer time of the reagent on this platform is very short. The simple chip design and fabrication are suitable for the development of commercial ultrafast PCR chips.