Nucleic Acid-based Amplification Research Articles

Highly sensitive detection of aflatoxin B1 byCRISPR/Cas12a-assisted single nanoparticle counting

CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated protein (Cas) have gained extensive applications in bioassays. However, CRISPR-based detection platforms are often hampered by limited analytical sensitivity, while nucleic acid-based amplification strategies are usually indispensable for additional signal enhancement with potential risks of amplification leakages. To address these challenges, an amplification-free CRISPR-based bioassay of aflatoxin B1 (AFB1) was proposed by applying single nanoparticle counting. Single-particle mode inductively coupled plasma mass spectrometry (Sp-ICPMS) has been regarded as a sensitive tool for nanoparticle counting since one nanoparticle can generate considerable signals above backgrounds. With AFB1, activator strands were introduced to initiate the trans-cleavage of CRISPR/Cas12a for cutting the nanoparticles-tagged-magnetic beads, which were transduced to nanoparticle count signals after separation. Finally, a pico-mole level limit-of-detections (LODs) with moderate selectivity was achieved. Certified reference materials (CRMs) analysis and recovery tests were conducted with promising results. To our best knowledge, this is the first report of the single particle counting-based CRISPR/Cas12a biosensing study.

Recent advances in optical biosensing and imaging of telomerase activity and relevant signal amplification strategies.

Telomerase as a new valuable biomarker for early diagnosis and prognosis evaluation of cancer has attracted much interest in the field of biosensors, cell imaging, and drug screening. In this review, we mainly focus on different optical techniques and various signal amplification strategies for telomerase activity determination. Fluorometric, colorimetry, chemiluminescence, surface-enhanced Raman scattering (SERS), and dual-mode techniques for telomerase sensing and imaging are summarized. Signal amplification strategies include two categories: one is nucleic acid-based amplification, such as rolling circle amplification (RCA), the hybridization chain reaction (HCR), and catalytic hairpin assembly (CHA); the other is nanomaterial-assisted amplification, including metal nanoclusters, quantum dots, transition metal compounds, graphene oxide, and DNA nanomaterials. Challenges and prospects are also discussed to provide new insights for future development of multifunctional strategies and techniques for in situ and in vivo analysis of biomarkers for accurate cancer diagnosis.

Highly reproducible electrochemical biosensor for Influenza A virus towards low-resource settings.

A highly reproducible electrochemical biosensor, employing a five-stranded four-way junction (5S-4WJ) system through square wave voltammetry, has been successfully validated for the detection of Influenza A virus (InfA). A comprehensive assessment of its linearity, precision, accuracy, and robustness has demonstrated its compliance with FDA standards. Integration with Nucleic Acid-Based Amplification (NASBA) has showcased its selectivity for InfA, enabling the detection of InfA RNA with a standard heater set at 41 °C. This platform offers a straightforward setup well-suited for use at low-resource facilities.

Rapid detection of viable Bacteroides in sewage-contaminated water using sodium dodecyl sulfate and propidium monoazide combined with loop-mediated isothermal amplification (LAMP) assay

Microbial source tracking using nucleic acid-based amplification techniques, including qPCR and LAMP is now routinely used to detect indicator bacteria in human faecal-contaminated water.

The temporal dynamics of Plasmodium species infection after artemisinin-based combination therapy (ACT) among asymptomatic children in the Hohoe municipality, Ghana

BackgroundThe routine surveillance of asymptomatic malaria using nucleic acid-based amplification tests is essential in obtaining reliable data that would inform malaria policy formulation and the implementation of appropriate control measures.MethodsIn this study, the prevalence rate and the dynamics of Plasmodium species among asymptomatic children (n = 1697) under 5 years from 30 communities within the Hohoe municipality in Ghana were determined.Results and discussionThe observed prevalence of Plasmodium parasite infection by polymerase chain reaction (PCR) was 33.6% (571/1697), which was significantly higher compared to that obtained by microscopy [26.6% (451/1697)] (P < 0.0001). Based on species-specific analysis by nested PCR, Plasmodium falciparum infection [33.6% (570/1697)] was dominant, with Plasmodium malariae, Plasmodium ovale and Plasmodium vivax infections accounting for 0.1% (1/1697), 0.0% (0/1697), and 0.0% (0/1697), respectively. The prevalence of P. falciparum infection among the 30 communities ranged from 0.0 to 82.5%. Following artesunate-amodiaquine (AS + AQ, 25 mg/kg) treatment of a sub-population of the participants (n = 184), there was a substantial reduction in Plasmodium parasite prevalence by 100% and 79.2% on day 7 based on microscopy and nested PCR analysis, respectively. However, there was an increase in parasite prevalence from day 14 to day 42, with a subsequent decline on day 70 by both microscopy and nested PCR. For parasite clearance rate analysis, we found a significant proportion of the participants harbouring residual Plasmodium parasites or parasite genomic DNA on day 1 [65.0% (13/20)], day 2 [65.0% (13/20)] and day 3 [60.0% (12/20)] after initiating treatment. Of note, gametocyte carriage among participants was low before and after treatment.ConclusionTaken together, the results indicate that a significant number of individuals could harbour residual Plasmodium parasites or parasite genomic DNA after treatment. The study demonstrates the importance of routine surveillance of asymptomatic malaria using sensitive nucleic acid-based amplification techniques.

A universal lateral flow assay for microRNA visual detection in urine samples

MicroRNAs (miRNAs) have been emerged as novel and significant biomarkers in liquid biopsy that can be found in different body fluids. Several techniques have been developed and applied for miRNAs analysis, including nucleic acid-based amplification methods, next generation sequencing, DNA microarrays and new genome-editing methods. These methods, however, are time-consuming and require expensive instruments and specially trained personnel. Biosensors, on the other hand, are alternative and valuable analytical/diagnostic tools due to their simplicity, cost-effectiveness, rapid analysis and ease of use. Several biosensors, especially nanotechnology-based ones, have been developed for miRNA analysis that are based either on target amplification or signal amplification and target re-cycling for sensitive detection. At this point of view, we have introduced a new and universal lateral flow assay in combination with reverse transcription – polymerase chain reaction (RT-PCR) and gold nanoparticles as reporters for the detection of miR-21 and miR-let-7a in human urine. It is the first time that such a biosensor has been applied to the detection of microRNAs in urine. As low as 102-103 copies of miR-21 and 102--104 copies of miR-let-7a added in urine were detectable by the proposed lateral flow assay with great specificity and repeatability (%CVs <4.5%).

Detection of Babesia bovis using loop-mediated isothermal amplification (LAMP) with improved thermostability, sensitivity and alternative visualization methods

Bovine babesiosis is one of the most economically important tick-borne diseases in tropical and subtropical countries. A conventional microscopic diagnosis is typically used because it is inexpensive and expeditious. However, it is highly dependent on well-trained microscopists and tends to be incapable of detecting subpatent and chronic infections. Here, we developed a novel nucleic acid-based amplification method using loop-mediated isothermal amplification (LAMP) in conjunction with a colori-fluorometric dual indicator for the rapid and accurate detection of Babesia bovis based on the mitochondrial cytochrome b gene. We aimed to improve the thermostability, sensitivity, specificity, and alternative visualization of LAMP-based methods. We assessed its diagnostic performance compared to two conventional PCR agarose gel electrophoresis (PCR-AGE) methods. The thermostability of LAMP reaction mixtures and DNA templates in variable conditions was also assessed. In addition, we evaluated alternative visualization methods using different light sources including neon, LED, and UV lights. We found that the LAMP-neon was ten times more sensitive than the PCR-AGE, while the LAMP-LED and LAMP-UV were 1,000 times more sensitive. The current LAMP method showed no cross-amplification with uninfected cattle DNA or other common blood parasites in cattle, including Babesia bigemina, Theileria orientalis, Anaplasma marginale, and Trypanosoma evansi. In addition, the developed LAMP method has good thermostability and the potential for on-site utility as B. bovis DNA could still be detected up to 72 h after initial preparation. Our findings suggested that the developed LAMP method provides an alternative approach for B. bovis detection with sensitivity higher than PCR-AGE diagnostics, high specificity, and the flexibility to use neon, LED, and UV light sources for positive signal observations.

Culture-Independent Raman Spectroscopic Identification of Bacterial Pathogens from Clinical Samples Using Deep Transfer Learning.

The rapid identification of bacterial pathogens in clinical samples like blood, urine, pus, and sputum is the need of the hour. Conventional bacterial identification methods like culturing and nucleic acid-based amplification have limitations like poor sensitivity, high cost, slow turnaround time, etc. Raman spectroscopy, a label-free and noninvasive technique, has overcome these drawbacks by providing rapid biochemical signatures from a single bacterium. Raman spectroscopy combined with chemometric methods has been used effectively to identify pathogens. However, a robust approach is needed to utilize Raman features for accurate classification while dealing with complex data sets such as spectra obtained from clinical isolates, showing high sample-to-sample heterogeneity. In this study, we have used Raman spectroscopy-based identification of pathogens from clinical isolates using a deep transfer learning approach at the single-cell level resolution. We have used the data-augmentation method to increase the volume of spectra needed for deep-learning analysis. Our ResNet model could specifically extract the spectral features of eight different pathogenic bacterial species with a 99.99% classification accuracy. The robustness of our model was validated on a set of blinded data sets, a mix of cultured and noncultured bacterial isolates of various origins and types. Our proposed ResNet model efficiently identified the pathogens from the blinded data set with high accuracy, providing a robust and rapid bacterial identification platform for clinical microbiology.

Recent advances in microchip electrophoresis for analysis of pathogenic bacteria and viruses.

Life-threatening diseases, such as hepatitis B, pneumonia, tuberculosis, and COVID-19, are widespread due to pathogenic bacteria and viruses. Therefore, the development of highly sensitive, rapid, portable, cost-effective, and selective methods for the analysis of such microorganisms is a great challenge. Microchip electrophoresis (ME) has been widely used in recent years for the analysis of bacterial and viral pathogens in biological and environmental samples owing to its portability, simplicity, cost-effectiveness, and rapid analysis. However, microbial enrichment and purification are critical steps for accurate and sensitive analysis of pathogenic bacteria and viruses in complex matrices. Therefore, we first discussed the advances in the sample preparation technologies associated with the accurate analysis of such microorganisms, especially the on-chip microfluidic-based sample preparations such as dielectrophoresis and microfluidic membrane filtration. Thereafter, we focused on the recent advances in the lab-on-a-chip electrophoretic analysis of pathogenic bacteria and viruses in different complex matrices. As the microbial analysis is mainly based on the analysis of nucleic acid of the microorganism, the integration of nucleic acid-based amplification techniques such as polymerase chain reaction (PCR), quantitative PCR, and multiplex PCR with ME will result in an accurate and sensitive analysis of microbial pathogens. Such analyses are very important for the point-of-care diagnosis of various infectious diseases.

Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV-2 Detection Using the HFman Probe.

Viral evolution impacts diagnostic test performance through the emergence of variants with sequences affecting the efficiency of primer binding. Such variants that evade detection by nucleic acid-based tests are subject to selective pressure, enabling them to spread more efficiently. Here, we report a variant-tolerant diagnostic test for SARS-CoV-2 using a loop-mediated isothermal nucleic acid-based amplification (LAMP) assay containing high-fidelity DNA polymerase and a high-fidelity DNA polymerase-medicated probe (HFman probe). In addition to demonstrating a high tolerance to variable SARS-CoV-2 viral sequences, the mechanism also overcomes frequently observed limitations of LAMP assays arising from non-specific amplification within multiplexed reactions performed in a single “pot”. Results showed excellent clinical performance (sensitivity 94.5%, specificity 100%, n = 190) when compared directly to a commercial gold standard reverse transcription quantitative polymerase chain reaction assay for the extracted RNA from nasopharyngeal samples and the capability of detecting a wide range of sequences containing at least alpha and delta variants. To further validate the test with no sample processing, directly from nasopharyngeal swabs, we also detected SARS-CoV-2 in positive clinical samples (n = 49), opening up the possibility for the assay’s use in decentralized testing.

SARS-CoV-2 RT-PCR positivity and antibody prevalence among asymptomatic hospital-based health care workers.

BackgroundThe level of asymptomatic infection with SARS-CoV-2 could be substantial and among health care workers (HCWs) a source of continuing transmission of the virus to patients and co-workers.ObjectivesMeasure the period prevalence of SARS-CoV-2 PCR positivity and seroprevalence of SARS-CoV-2 IgG antibodies among a random sample of asymptomatic health system hospital-based health care workers (HCWs) 6½ -15½ weeks after 4/5/2020, the peak of the first surge of COVID-19 admissions.ResultsOf 524 eligible and consented participants from four metropolitan hospitals, nasopharyngeal swabs were obtained from 439 (83.8 %) and blood from 374 (71.4 %). Using PCR nucleic acid-based amplification (NAAT) methods, the period prevalence of SARS-CoV-2 infection was 0.23 % (95 % confidence interval (CI) 0.01 %–1.28 %; 1/439) from 5/21/20−7/16/20. The seroprevalence of SARS-CoV-2 IgG antibodies from June 17-July 24, 2020 was 2.41 % (95 % CI 1.27 %–4.51 %; 9/374). Those who were reactive were younger (median age 36 versus 44 years; p = 0.050), and those with self-reported Hispanic/Latino ethnicity had a higher seroprevalence (2/12 = 16.7 % versus 7/352 = 2.0 %; p = 0.051). There were no significant differences by sex, race, residence, hospital, unit or job type. The one employee who was found to be PCR test positive in this study was also reactive for IgG antibodies, tested 27 days later.ConclusionsThe period prevalence of PCR positivity to SARS-CoV-2 and IgG seroprevalence was unexpectedly low in asymptomatic HCWs after a peak in COVID-19 admissions and the establishment of state and institutional infection control policies, suggesting that routine screening tests while community prevalence is relatively low would produce a minimal yield.

Whole genome characterization of strains belonging to the Ralstonia solanacearum species complex and in silico analysis of TaqMan assays for detection in this heterogenous species complex

Identification and classification of members of the Ralstonia solanacearum species complex (RSSC) is challenging due to the heterogeneity of this complex. Whole genome sequence data of 225 strains were used to classify strains based on average nucleotide identity (ANI) and multilocus sequence analysis (MLSA). Based on the ANI score (>95%), 191 out of 192(99.5%) RSSC strains could be grouped into the three species R. solanacearum, R. pseudosolanacearum, and R. syzygii, and into the four phylotypes within the RSSC (I,II, III, and IV). R. solanacearum phylotype II could be split in two groups (IIA and IIB), from which IIB clustered in three subgroups (IIBa, IIBb and IIBc). This division by ANI was in accordance with MLSA. The IIB subgroups found by ANI and MLSA also differed in the number of SNPs in the primer and probe sites of various assays. An in-silico analysis of eight TaqMan and 11 conventional PCR assays was performed using the whole genome sequences. Based on this analysis several cases of potential false positives or false negatives can be expected upon the use of these assays for their intended target organisms. Two TaqMan assays and two PCR assays targeting the 16S rDNA sequence should be able to detect all phylotypes of the RSSC. We conclude that the increasing availability of whole genome sequences is not only useful for classification of strains, but also shows potential for selection and evaluation of clade specific nucleic acid-based amplification methods within the RSSC.

Multi-channel collection of G-quadruplex transducers for amplified signaling of Pax-5 based on target-triggered split-to-intact remodeling of dual-G-rich duplex probe

Nucleic acids-sensing methods provide the potential to enable and hasten early disease detection and to rationalize treatments, especially for cancers. In this work, a colorimetric label-free sensing system based on target-triggered split-to-intact remodeling of dual-G-rich duplex probe (Dual-GDP) has been developed for amplified signaling of Pax-5 gene. In this sensing system, the Dual-GDP is splited into one hairpin-structured G-rich probe (HGP) and one duplex-shaped G-rich probe (DGP), which can be re-built to an intact form through target gene triggered accurate base-pairing. Significantly, this unique split-to-intact remodeling behavior allows the occurrence of a well-designed active isothermal amplification that can amplify target response with seven stages to multi-channel accumulate numbers of G-quadruplex transducers. When specifically combined with hemin, the formed G-quadruplex-hemin complexes, also known as DNAzymes, can strongly catalyze the oxidization of TMB to a colored TMB+ and robustly reflect the target gene even in complex biological environments. We demonstrate that this method offers simplified operations and improved assay abilities for Pax5 gene analysis. We envision these findings might provide new insights to exploit versatile oligonucleotide probes and nucleic acid-based amplification strategies for diverse bioanalytical applications.

Review of Electrochemical DNA Biosensors for Detecting Food Borne Pathogens.

The vital importance of rapid and accurate detection of food borne pathogens has driven the development of biosensor to prevent food borne illness outbreaks. Electrochemical DNA biosensors offer such merits as rapid response, high sensitivity, low cost, and ease of use. This review covers the following three aspects: food borne pathogens and conventional detection methods, the design and fabrication of electrochemical DNA biosensors and several techniques for improving sensitivity of biosensors. We highlight the main bioreceptors and immobilizing methods on sensing interface, electrochemical techniques, electrochemical indicators, nanotechnology, and nucleic acid-based amplification. Finally, in view of the existing shortcomings of electrochemical DNA biosensors in the field of food borne pathogen detection, we also predict and prospect future research focuses from the following five aspects: specific bioreceptors (improving specificity), nanomaterials (enhancing sensitivity), microfluidic chip technology (realizing automate operation), paper-based biosensors (reducing detection cost), and smartphones or other mobile devices (simplifying signal reading devices).

NaYF4:Yb,Er Upconversion Nanotransducer with in Situ Fabrication of Ag2S for Near-Infrared Light Responsive Photoelectrochemical Biosensor.

An innovative near-infrared (NIR) light-driven photoelectrochemical (PEC) aptasensor was constructed for sensitive screening of carcinoembryonic antigen (CEA) on the basis of in situ formation of Ag2S nanoparticles on the NaYF4:Yb,Er upconversion nanoparticles (UCNs), coupled with hybridization chain reaction (HCR) for the signal amplification. Utilization of UCN as the light nanotransducer could convert the NIR light into an applicable wavelength harvested by semiconductors. The multiemissions of NaYF4:Yb,Er UCN could match well with the absorption characteristics of Ag2S. In the presence of target CEA, a sandwich-type reaction was carried out between capture CEA aptamer/NaYF4:Yb,Er-modified electrode and trigger CEA aptamer, which underwent an unbiased strand-displacement reaction to open C-rich hairpin probes in sequence between two alternating hairpins with the assistance of C-Ag+-C chelation reaction. Upon addition of sulfide, the chelated Ag+ ions in the long-nicked DNA poly strands by hybridization chain reaction reacted with sulfide to generate Ag2S nanoparticles. The formed Ag2S could utilize effectively the upconversion emissions to amplify the photocurrent. Under optimal conditions, NaYF4:Yb,Er-based NIR light-responsive PEC aptasensing platform exhibited high sensitivity for the determination of CEA within a dynamic linear range of 0.005-5.0 ng mL-1. The limit of detection was 1.9 pg mL-1. Good precision and high specificity could be acquired in this system for the analysis of target CEA. Human serum samples containing target CEA were measured using our strategy and received well-matched results relative to human CEA enzyme-linked immunosorbent assay kits. Importantly, the NaYF4:Yb,Er-based NIR light-responsive PEC aptasensing system provides a new ideal for the detection of disease-related biomarkers using a nucleic acid-based amplification strategy.

Nanoparticle Counting by Microscopic Digital Detection: Selective Quantitative Analysis of Exosomes via Surface-Anchored Nucleic Acid Amplification.

Exosomes are nanosized vesicles secreted by cells, with a lipid bilayer membrane and protein and nucleic acid contents. Here, we present the first method for the selective and quantitative analysis of exosomes by digital detection integrated with nucleic acid-based amplification in a microchip. An external biocompatible anchor molecule conjugated with DNA oligonucleotides was anchored in the lipid bilayer membrane of exosomes via surface self-assembly for total exosome analysis. Then, specific antibody-DNA conjugates were applied to label selective exosomes among the total exosomes. The DNA-anchored exosomes were distributed into microchip chambers with one or fewer exosomes per chamber. The signal from the DNA on the exosomes was amplified by a rapid isothermal nucleic acid detection assay. A chamber with an exosome exhibited a positive signal and was recorded as 1, while a chamber without an exosome presented a negative signal and was recorded as 0. The 10100101 digital signals give the number of positive chambers. According to the Poisson distribution, the exosome stock concentration was calculated by the observed fraction of positive chambers. The findings showed that nanoscale particles can be digitally detected via DNA-mediated signal amplification in a microchip with simple microscopic settings. This approach can be integrated with multiple types of established nucleic acid assays and provides a versatile platform for the quantitative detection of various nanosomes, from extracellular vesicles such as exosomes and enveloped viruses to inorganic and organic nanoparticles, and it is expected to have broad applications in basic research areas as well as disease diagnosis and therapy.

Laboratory Impact of Rapid Molecular Tests used for the Detection of Respiratory Pathogens

With outbreaks of new respiratory viruses such as the severe acute respiratory syndrome coronavirus and swine-origin influenza A/H1N1, the nucleic acid-based amplification test was introduced to identify causative agents. Multiplex PCR, which can simultaneously detect various respiratory pathogens, is currently used worldwide. Recently, a new type of multiplexed molecular test using a fully automated workflow system was developed, which was also adapted to our laboratory. In this study, we assessed improvements in laboratory practice brought about by the implementation of the rapid test for the detection of respiratory viruses. We investigated the number of routine and rapid tests conducted as well as the change in monthly test frequency of the routine test. We also analyzed the waiting time, turnaround time, and lead time for the routine and rapid tests. The Anyplex II RV16 detection kit (Seegene, Seoul, Korea) and Filmarray Respiratory Panel (BioFire Diagnostics, Inc., Salt Lake City, UT, USA) were used for the routine and rapid tests, respectively. Compared to the routine test, the rapid test significantly (p < 0.01) decreased the mean waiting time (1 hour 46 minutes), turnaround time (1 hour 45 minutes), and lead time (3 hours 32 minutes). After the implementation of the rapid test, the number of routine tests conducted was reduced over the 5-month period, from 13 times a month to 3 times a month. The implementation of the rapid test for the detection of respiratory viruses improved the diagnostic efficiency of the laboratory and greatly reduced lead time.

Gold nanoparticle enhanced surface plasmon resonance imaging of microRNA-155 using a functional nucleic acid-based amplification machine

MicroRNAs (miRNAs) are considered as being promising biomarkers for hematological malignancies, their aging, progression and prognosis. The authors have developed a method for the detection of miRNA-155 by using surface plasmon resonance (SPR) imaging coupled to a nucleic acid-based amplification strategy using gold nanoparticles (AuNPs). The target miRNA-155 is captured by surface-bound DNA probes. After hybridization, DNA-AuNP are employed for signal amplification via DNA sandwich assembly, resulting in a large increase in the SPR signal. This method can detect miRNA-155 in concentrations down to 45 pM and over dynamic that extends from 50 pM to 5 nM. The assay is highly specific and can discriminate even a single base mismatch. It also is reproducible, precise, and was successfully applied to the determination of miRNA-155 in spiked real samples where it gave recoveries in the range between 86% and 98%. This biosensor provides an alternative approach for miRNA detection in biomedical research and clinical diagnosis, which is highly effective and efficient.

Research Article Comparison Between Real-Time Polymerase Chain Reaction and DNA-Microarray in Detection and Identification of Mycobacterium Species

Low-cost and low-density (LCD) DNA arrays offer an easy way to detect resistance as minimal laboratory instrumentation is needed. Nucleic acid-based amplification tests allow the rapid detection of Mycobacterium tuberculosis. Recently, a real-time PCR assay for M. tuberculosis complex was introduced. Real-Time PCR and DNA-microarray techniques were compared with the classical methods of Ziehl-Neelsen (ZN) staining and culturing. Regarding to the standard culture method, 80 positive individuals were identified out of 140 urine samples. RT- PCR showed 96.3% sensitivity and 96.7% specificity with Mycobacterial tuberculosis complex (MTB) (n=10) and nontuberculous mycobacteria (NTM) (n=70). The DNA-microarray analysis exhibited 100% sensitivity and specificity. One species belonging to (MTB) was identified as M. tuberculosis and positively represented by 12.5% (n=10). Five species belonging to nontuberculous mycobacteria (NTM) were identified and represented as M. kansasii 37.5% (n=30), M. celatum 21.25% (n=17), M. gordonae 11.25% (n=9), M. chelonae 10% (n=8), and M. phlei 7.5% (n=6). The results recommend the use of our simple and rapid PCR technique for early diagnosis of mycobacteria’s. Also, the fast LCD-microarray protocol is very useful for species identification and differentiation between MTB and NTM

Influenza A virus drift variants reduced the detection sensitivity of a commercial multiplex nucleic acid amplification assay in the season 2014/15.

The influenza season 2014/15 was dominated by drift variants of influenza A(H3N2), which resulted in a reduced vaccine effectiveness. It was not clear if the performance of commercial nucleic-acid-based amplification (NAT) assays for the detection of influenza was affected. The purpose of this study was to perform a real-life evaluation of two commercial NAT assays. During January-April 2015, we tested a total of 665 samples from patients with influenza-like illness using the Fast Track Diagnostics Respiratory pathogens 21, a commercial multiplex kit, (cohorts 1 and 2, n=563 patients) and the Xpert Flu/RSV XC assay (cohort 3, n=102 patients), a single-use cartridge system. An in-house influenza real-time RT-PCR (cohort 1) and the RealStar Influenza RT-PCR 1.0 Kit (cohort 2 and 3) served as reference tests. Compared to the reference assay, an overall agreement of 95.9% (cohort 1), 95% (cohort 2), and 98% (cohort 3) was achieved. A total of 24 false-negative results were observed using the Fast Track Diagnostics Respiratory pathogens 21 kit. No false-negative results occurred using the Xpert Flu/RSV XC assay. The Fast Track Diagnostics Respiratory pathogens 21 kit and the Xpert Flu/RSV XC assay had sensitivities of 90.7% and 100% and specificities of 100% and 94.1%, respectively, compared to the RealStar 1.0 kit. Upon modification of the Fast Track Diagnostics Respiratory pathogens 21 kit, the sensitivity increased to 97.3%. Influenza virus strains circulating during the 2014/15 season reduced the detection sensitivity of a commercial NAT assay, and continuous monitoring of test performance is therefore necessary.