Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection.

The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid-based tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the U.S. Centers for Disease Control and Prevention takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply, and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the "gold-standard" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own laboratories. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.

The selection and identification of compound housekeeping genes for quantitative real-time polymerase chain reaction analysis in rat fetal kidney.

During quantitative real-time polymerase chain reaction (RT-qPCR) data analysis, the selection of optimal housekeeping gene is necessary to ensure the accuracy of results. It is noteworthy that housekeeping genes commonly used in adult studies may not be applicable for fetus. However, the stability analysis of housekeeping gene in fetal kidney has not been reported. This study intends to screen the applicable compound housekeeping genes in rat fetal kidney. In this study, eight housekeeping genes used in kidney studies based on literature reports (GAPDH, ACTB, 18S, HPRT, YWHAZ, HMBS, PPIA, and TBP) were selected as the research object. Their expression levels in the rat fetal kidney in physiological condition and the intrauterine growth retardation (IUGR) model induced by prenatal dexamethasone exposure (PDE) (0.2 mg/kg·day from gestation Days 9 to 20) was measured. Furthermore, these eight housekeeping genes were used to conduct relative quantitative analysis of nephrin expression in the fetal kidney in PDE-induced IUGR model, to compare the influence of choosing different housekeeping gene on data analysis of nephrin expression and to verify the reliability of selected compound housekeeping genes. In this study, stable housekeeping genes of fetal kidney tissues in PDE-induced IUGR model were identified: ACTB, GAPDH, TBP, and HMBS for males; ACTB, YWHAZ, and GAPDH for females. Besides, our results suggest that ACTB + GAPDH were the best compound housekeeping genes for normalization analysis in male fetal kidney studies, and ACTB + YWHAZ in females. This study will provide an experimental evidence basis for the selection of housekeeping genes in the RT-qPCR experiment in renal development toxicology-related models.

Detection of dengue, chikungunya, and Zika RNA in blood donors from Southeast Asia.

BACKGROUND: Chikungunya (CHIKV), dengue (DENV), and Zika (ZIKV) viruses are of concern due to the potential of transfusion transmission in blood, especially in regions such as Southeast Asia where the viruses are endemic. The recent availability of nucleic acid testing (NAT) to screen blood donations on an automated platform provides the opportunity to detect potentially infectious units in asymptomatic donors. STUDY DESIGN AND METHODS: Three thousand blood donations from Vietnam and 6000 from Thailand were screened with a real-time polymerase chain reaction (PCR) test (cobas CHIKV/DENV, Roche Diagnostics, Indianapolis, IN) and equal numbers on cobas Zika (Roche Diagnostics). Reactive samples were tested by alternative NAT with resolution of discordant results by heminested PCR. Throughput of simultaneous testing of the two assays on the cobas 8800 system (Roche Diagnostics) was evaluated. RESULTS: In Vietnam, 9 of 3045 samples were reactive for DENV and all were confirmed, for a prevalence (with 95% confidence interval [CI]) of 0.296% (0.135-0.560). In Thailand, 2 of 6000 samples were reactive for CHIKV, 4 of 6000 for DENV, and 1 of 6005 for ZIKV, and all confirmed. The prevalence of CHIKV is 0.033% (0.004-0.120), DENV 0.067% (0.018-0.171), and ZIKV 0.017% (0.000-0.093). The overall specificity for the cobas CHIKV/DENV and cobas Zika tests was 100% (99.959-100). For the simultaneous assay testing, 960 test results were available in 7 hours and 53 minutes. CONCLUSION: Detection of CHIKV, DENV, and ZIKV RNA in donor samples in Vietnam and Thailand indicate the presence of the virus in asymptomatic blood donors. The cobas 6800/8800 systems (Roche Molecular Systems, Pleasanton, CA) enable screening blood donations in endemic areas for these viruses together or separately.

Comparison between Abbott m2000 RealTime and Alinity m STI systems for detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium.

The new Abbott Alinity m STI Assay was compared with Abbott m2000 RealTime PCR. For Chlamydia trachomatis, 26 (7.5%) of 347 samples were positive in the Alinity assay and 24 (6.9%) in the m2000 assay. Corresponding figures for Neisseria gonorrhoeae were 23 (6.6%) and 17 (4.9%). For Mycoplasma genitalium, 22 (7.9%) of 279 samples were positive in the Alinity assay and 18 (6.5%) in the m2000 assay, for which DNA extraction was performed on an m2000sp instrument combined with in-house real-time PCR. The Alinity assay has at least the same sensitivity as the m2000 assay. The specificity was evaluated by discrepancy analysis.

Ultrasensitive monitoring strategy of PCR-like levels for zearalenone contamination based DNA barcode.

BACKGROUND: The ultrasensitive monitoring strategy of zearalenone (ZEN) is essential and desirable for food safety and human health. In the present study, a coupling of gold nanoparticles-DNA barcode and direct competitive immunoassay-based real-time polymerase chain reaction signal amplification (RT-IPCR) for ZEN close to the sensitivity of PCR-like levels is described and evaluated. RESULTS: The RT-IPCR benefited from the use of a DNA barcode and RT-PCR detection strategy, thus resulting in ultrasensitive and simple detection for ZEN. Under the optimal RT-IPCR, the linear range of detection was from 0.5 to 1000 pg mL-1 and the limit of detection was 0.5 pg mL-1 , which was 400-fold lower than the enzyme-linked immunosorbent assay. The detection procedure was simplified and the detection time was shortened. The specificity, accuracy and precision of the RT-IPCR confirmed a high performance. ZEN-positive contamination levels were from 0.056 to 152.12 ng g-1 by the RT-IPCR, which was demonstrated to be highly reliable by liquid chromatography-tandem mass spectrometry. CONCLUSION: The proposed RT-IPCR could be used as an alternative for detecting ZEN with satisfactory ultrasensitivity, simplicity, low cost and high-throughput. The present study could provide a strategy for the ultrasensitive detection of the small molecule with a simple and practical approach, which has significant appeal and application prospects.

Detection of aberrant methylation of HOXA9 and HIC1 through multiplex MethyLight assay in serum DNA for the early detection of epithelial ovarian cancer.

Accumulated evidence revealed that aberrant CpG island hypermethylation plays an important role in carcinogenesis which can serve as a promising target for molecular detection in body fluids. Despite a myriad of attempts to diagnose ovarian cancer (OC) at an early stage, this clinical aim remains a major challenge. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant DNA methylation patterns in circulating DNA provide highly specific cancer signals. In our study, we establish a novel panel of methylation-specific genes for the development of a TaqMan based qPCR assay to quantify methylation levels. We analyzed promoter methylation of homeobox A9 (HOXA9) and hypermethylated in cancer 1 (HIC1) quantitatively in 120 tissue samples and in 70 matched serum cell-free DNA (CFDNA) of cancerous and noncancerous samples by MethyLight assay. HOXA9 and HIC1 methylation occurred in 82.3 and 80.0% of OC tissue samples in singleplex assay, thereby confirming that methylation was highly cancer-specific. When either or both gene promoter showed methylation, the sensitivity was 88.2% with a specificity of 88.6% in tissue samples. The combined sensitivity for this novel marker panel in serum CFDNA was 88.9% (area under the curve [AUC] = 0.95). In contrast, no hypermethylation was observed in serum from matched cancer-free control women. Our results confirm the elevated performance of novel epigenetic marker panel (HOXA9 and HIC1) when analyzed in tissue and matched serum samples. Our findings reveal the potential of this biomarker panel as a suitable diagnostic serum biomarker for early screening of OC.

A handheld continuous-flow real-time fluorescence qPCR system with a PVC microreactor.

The polymerase chain reaction (PCR) has unique advantages of sensitivity, specificity and rapidity in pathogen detection, which makes it at the forefront of academia and application in molecular biology diagnosis. In this study, we proposed a hand-held real-time fluorescence qPCR system, which can be used for the quantitative analysis of nucleic acid molecules. For the first time, we use a PVC microreactor which improved the transmittance of the microreactor and made it easy to collect the fluorescence signal. In order to make it portable, the system adopted a passive syringe for sample injection and integrated temperature control and detection with a lithium battery for power supply. What's more, the fluorescence signal was captured by using a smartphone through an external automatic robotic arm. This real-time qPCR system can detect genomic DNA of the H7N9 avian influenza over four orders of magnitude of concentration from 107 to 104 copies per µL. In addition, it was verified that the fluorescence images obtained by this system were clearer than those obtained by a traditional system (using a PTFE spatial PCR microreactor) with two typical dyes and a probe tested-EvaGreen, SYBR Green and FAM.

Comparison of paired cerebrospinal fluid and serum cell-free mitochondrial and nuclear DNA with copy number and fragment length.

BACKGROUND: Most studies on cell-free DNA (cfDNA) were only for single body fluids; however, the differences in cfDNA distribution between two body fluids are rarely reported. Hence, in this work, we compared the differences in cfDNA distribution between cerebrospinal fluid (CSF) and serum of patients with brain-related diseases. METHODS: The fragment length of cfDNA was determined by using Agilent 2100 Bioanalyzer. The copy numbers of cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) were determined by using real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) with three pairs of mitochondrial ND1 and nuclear GAPDH primers, respectively. RESULTS: There were short (~60 bp), medium (~167 bp), and long (>250 bp) cfDNA fragment length distributions totally obtained from CSF and serum using Agilent 2100 Bioanalyzer. The results of both qPCR and ddPCR confirmed the existence of these three cfDNA fragment ranges in CSF and serum. According to qPCR, the copy numbers of long cf-mtDNA, medium, and long cf-nDNA in CSF were significantly higher than in paired serum. In CSF, only long cf-mtDNA's copy numbers were higher than long cf-nDNA. But in serum, the copy numbers of medium and long cf-mtDNA were higher than the corresponding cf-nDNA. CONCLUSION: The cf-nDNA and cf-mtDNA with different fragment lengths differentially distributed in the CSF and serum of patients with brain disorders, which might serve as a biomarker of human brain diseases.

Efficacy of alcohol-based hand sanitizers against human norovirus using RNase-RT-qPCR with validation by human intestinal enteroid replication.

Successful human norovirus (HuNoV) cultivation in stem cell-derived human intestinal enteroids (HIE) was recently reported. The purpose of this study was to evaluate the anti-HuNoV efficacy of two alcohol-based commercial hand sanitizers and 60% ethanol by suspension assay using RNase-RT-qPCR, with subsequent validation of efficacy by HuNoV cultivation using the HIE model. In suspension, when evaluated by RNase-RT-qPCR, 60% ethanol resulted in less than one log10 reduction in HuNoV genome equivalent copies (GEC) regardless of contact time (30 or 60s) or soil load. The two commercial products outperformed 60% ethanol regardless of contact time or soil load, providing 2·2-3·2 log10 HuNoV GEC reductions by suspension assay. Product B could not be validated in the HIE model due to cytotoxicity. Following a 60s exposure, viral replication in the HIE model increased 1·9 ± 0·2 log10 HuNoV GEC for the neutralization (positive) control and increased 0·9 ± 0·2 log10 HuNoV GEC in challenged HIE after treatment with 60% ethanol. No HuNoV replication in HIE was observed after a 60 s exposure to Product A.

A TaqMan probe based real-time PCR for the detection of Decapod iridescent virus 1.

Decapod iridescent virus 1 (DIV1) was proven to be the aetiological agent of a disease causing mass die-offs of shrimp, prawn and crayfish. The specific purpose of this study was to develop a new sensitive real-time PCR method for the specific detection of DIV1. A pair of primers that amplify a 142 bp fragment and a TaqMan probe were selected for the major capsid protein gene of DIV1. They were shown to be specific for DIV1 and did not react with other common shrimp pathogens or healthy shrimp DNA. The method could detect as virus levels as low as 1.2 copies of DIV1 plasmid DNA.

Multicentre comparison of quantitative PCR-based assays to detect SARS-CoV-2, Germany, March 2020.

Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we compare 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic.

Viability Quantitative PCR Utilizing Propidium Monoazide, Spheroplast Formation, and Campylobacter coli as a Bacterial Model.

A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen Campylobacter coli as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA into dead cells, and exploitation of an internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of C. coli cells by optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 to 8.5 log10 viable cells ml-1), and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media, and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable-but-nonculturable state and might be utilized for the quantification of other Gram-negative bacteria.IMPORTANCE There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable-but-nonculturable state. The high sensitivity and specificity of the quantitative PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into dead Campylobacter coli cells via spheroplasting and in developing an internal sample process control, thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens.

A fully portable microchip real-time polymerase chain reaction for rapid detection of pathogen.

Point-of-care detection for pathogen is of critical need for wide epidemic warning and medical diagnosis. In this work, we have designed and developed a fully portable and integrated microchip based real-time polymerase chain reaction machine for rapid pathogen detection. The instrument consists of three functional components including heating, optical, and electrical modules, which are integrated into a portable compact box. The microchip is consumable material replaceable to meet various detection needs. Consequently, we demonstrated the outstanding performance of this portable machine for rapid detection of Salmonella and Escherichia coli O157:H7 with the advantage of time-saving (∼25 min), less samples consumption, portability, and user-friendly operation.

EWOD silicon biosensor for multiple nucleic acids analysis.

In this paper, a miniaturized biosensor containing 96 silicon microchambers electroloaded with nano-volumes of liquid (EW-chip) is presented. The liquid electroloading is achieved by the appropriate modulation of interface properties. The surface chemistries have been studied to guarantee effective interface properties for both electrowetting on dielectric actuation and biocompatibility versus biochemical reactions. The silicon microchambers are 200 nl in volume and are connected to a specific system of electrodes able to deliver liquid sample on each well. The device also integrates temperature sensors and heaters to perform biochemical reactions. On that, the effectiveness of this device has been successfully proven towards the nucleic acids detection via real-time polymerase chain reaction amplification. Hepatitis B virus genome target has been used to assess the device performance. Results show very uniform amplification over the 96 microchambers without any cross-contamination process. These features make this system a very appealing potential solution for genetic point-of-care devices where a high level of parallelism of analysis is required.

Development of the MitoQ assay as a real-time quantification of mitochondrial DNA in degraded samples.

Mitochondrial DNA is a reliable genetic material for estimating maternally related haplogroups and ancestries. Exploring maternal DNA inheritance is particularly useful when nuclear DNA is degraded or limited, as the copy number of mitochondrial DNA is far greater than the copy number of nuclear DNA. Normal mitochondrial DNA copy number has been estimated to 100 copies per buccal epithelial cell, 4000 copies in skeletal cells and 7000 copies in myocardial cells. This estimation is usually performed via extrapolation from the nuclear DNA quantitation. It is essential to reduce this variability and accurately quantify the exact number of copies of mitochondrial DNA, especially in compromised samples of a forensic or ancient nature. While useful, the testing of mitochondrial DNA is often long and costly and comes with limited success. The accurate quantification of mitochondrial DNA using specific quantitative PCR assays can be used to make better decisions on the downstream testing and success of amplification. As a result, this study develops a real-time assay for the quantification of mitochondrial DNA copy number and assesses its performance on a set of degraded DNA samples. The developed MitoQ assay has been shown to be highly specific to the human mitochondrial genome with no amplification of nuclear pseudogenes being observed and outperformed a previously published concordant assay. Additionally, a high sensitivity was measured to 280 copies of mitochondrial DNA. Minimal variation was observed between each replication cycle, indicating the assay to be robust and repeatable. Overall, this study presents a real-time assay that is sensitive and robust to quantifying mitochondrial DNA copy number in degraded samples. Furthermore, there is potential to incorporate the assay as an additional target in current qPCR assays which use a six-dye chemistry and provide a complete overview of a sample's quality and quantity.

An innovative silicon-chip for sensitive real time PCR improvement in pathogen detection.

An innovative miniaturized silicon-chip was developed for highly sensitive detection of pathogen genomes of both viruses and bacteria through real time PCR (qRT-PCR). The device was properly designed to enhance the optical signal and perform accurate thermal control. Results show an improvement of PCR amplification by one order of magnitude in sensitivity compared to the standard RT-PCR method. In particular for hepatitis B virus a decrease of the mean value of Ct of about 2.9 ± 0.9 compared to the standard system was observed. Similarly, for the bacteria Pseudomonas aeruginosa, Staphylococcus aureus and Acinetobacter baumannii, a decrease of the mean values of Ct of 1.8 ± 0.5, 3.1 ± 0.5 and 3.9 ± 0.9, respectively, was observed.

Development of an Inexpensive and Rapid Operation Device for High-Throughput Real-Time Quantitative PCR-Based CYP2D6 CNV Genotyping.

The CYP2D6 gene is the most well characterized gene involved in drug metabolism and is known to have both gene duplication and deletion variants. We report an optimized method for the determination of copy number variation (CNV) in the CYP2D6 gene by a novel purification process for a real-time quantitative PCR. This high-throughput low-cost method accurately determines CNV in the CYP2D6 gene enabling reliable estimates of disease prediction in large epidemiological samples.

Development of Mobile Laboratory for Viral Hemorrhagic Fever Detection in Africa.

Background: A mobile laboratory transportable on commercial flights was developed to enable local response to viral hemorrhagic fever outbreaks. Methods: The development progressed from use of mobile real-time reverse-transcription polymerase chain reaction to mobile real-time recombinase polymerase amplification. In this study, we describe various stages of the mobile laboratory development. Results: A brief overview of mobile laboratory deployments, which culminated in the first on-site detection of Ebola virus disease (EVD) in March 2014, and their successful use in a campaign to roll back EVD cases in Conakry in the West Africa Ebola virus outbreak are described. Conclusions: The developed mobile laboratory successfully enabled local teams to perform rapid disgnostic testing for viral hemorrhagic fever.

An Affordable and Portable Thermocycler for Real-Time PCR Made of 3D-Printed Parts and Off-the-Shelf Electronics.

The polymerase chain reaction (PCR) is a sought-after nucleic acid amplification technique used in the detection of several diseases. However, one of the main limitations of this and other nucleic acid amplification assays is the complexity, size, maintenance, and cost of their operational instrumentation. This limits the use of PCR applications in settings that cannot afford the instruments but that may have access to basic electrical, electronic, and optical components and the expertise to build them. To provide a more accessible platform, we developed a low-cost, palm-size, and portable instrument to perform real-time PCR (qPCR). The thermocycler leverages a copper-sheathed power resistor and a computer fan, in tandem with basic electronic components controlled from a single-board computer. The instrument incorporates a 3D-printed chassis and a custom-made fluorescence optical setup based on a CMOS camera and a blue LED. Results are displayed in real-time on a tablet. We also fabricated simple acrylic microdevices consisting of four wells (2 µL in volume each) where PCR reactions take place. To test our instrument, we performed qPCR on a series of cDNA dilutions spanning 4 orders of magnitude, achieving similar limits of detection as those achieved by a benchtop thermocycler. We envision our instrument being utilized to enable routine monitoring and diagnosis of certain diseases in low-resource areas.

The Brazilian experience of nucleic acid testing to detect human immunodeficiency virus, hepatitis C virus, and hepatitis B virus infections in blood donors.

BACKGROUND: The history of the development and implementation of the Brazilian nucleic acid testing (NAT) platform to detect and discriminate among human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections in blood donors is described here. The results for the sensitivity, reproducibility, and NAT yield of the platform since program implementation are provided. STUDY DESIGN AND METHODS: The Brazilian NAT HIV, HCV, and HBV kit was developed and evaluated with regard to analytical sensitivity, specificity, intralot and interlot reproducibility, interfering substances, and genotype and diagnostic sensitivity. Additionally, a sample of identified NAT-yield cases was characterized with regard to viral load. RESULTS: The 95% limits of detection for HIV, HCV, and HBV were 68.02, 102.35, and 9.08 IU/mL, respectively. All replicates were detected with reproducibility assays between the acceptable values. A total of 13,610,536 blood donors was screened from 2010 to 2016, and 63 HIV-yield cases and 28 HCV-yield cases were detected. Among 5,795,424 blood donors screened for HBV from 2014 to 2016, 42 yield cases were found. CONCLUSION: The Brazilian NAT HIV, HCV, and HBV kit is an automated NAT system suitable for routine blood donor screening in a completely traceable process. The analytical sensitivity as well as the diagnostic sensitivity fulfilled all requirements set by the health ministry for blood donor screening. A significant number of transmission cases were prevented by the implementation of this important program.