An ultra-portable, self-contained point-of-care nucleic acid amplification test for diagnosis of active COVID-19 infection.
Ontology highlight
ABSTRACT:
SUBMITTER: Deng H
PROVIDER: S-EPMC8313664 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting.
| S-EPMC7349311 | biostudies-literature
Project description:The Mycobacterium tuberculosis complex (MTBC) remains one of the top 10 leading causes of death globally. The early diagnosis of MTBC can reduce mortality and mitigate disease transmission. However, current nucleic acid amplification diagnostic test methods are generally time-consuming and show suboptimal diagnostic performance, especially in extrapulmonary MTBC samples or acid-fast stain (AFS)-negative cases. Thus, development of an accurate assay for the diagnosis of MTBC is necessary, particularly under the above mentioned conditions. In this study, a single-tube nested real-time PCR assay (N-RTP) was developed and compared with a newly in-house-developed high-sensitivity real-time PCR assay (HS-RTP) using 134 clinical specimens (including 73 pulmonary and 61 extrapulmonary specimens). The amplification efficiency of HS-RTP and N-RTP was 99.8% and 100.7%, respectively. The sensitivity and specificity of HS-RTP and N-RTP for the diagnosis of MTBC in these specimens were 97.5% (77/79) versus 94.9% (75/79) and 80.0% (44/55) versus 89.1% (49/55), respectively. The sensitivity and specificity of HS-RTP and N-RTP for the diagnosis of MTBC in pulmonary specimens were 96.3% (52/54) versus 96.3% (52/54) and 73.7.0% (14/19) versus 89.5% (17/19), respectively; in extrapulmonary specimens, the sensitivity and specificity of HS-RTP and N-RTP were 100% (25/25) versus 92% (23/25) and 83.3% (30/36) versus 88.9% (32/36), respectively. Among the AFS-negative cases, the sensitivity and specificity of HS-RTP and N-RTP were 97.0% (32/33) versus 90.9% (30/33) and 88.0% (44/50) versus 92.0% (46/50), respectively. Overall, the sensitivity of HS-RTP was higher than that of N-RTP, and the performance was not compromised in extrapulmonary specimens and under AFS-negative conditions. In contrast, the specificity of the N-RTP assay was higher than that of the HS-RTP assay in all types of specimens. In conclusion, the HS-RTP assay would be useful for screening patients suspected of exhibiting an MTBC infection due to its higher sensitivity, while the N-RTP assay could be used for confirmation because of its higher specificity. Our results provide a two-step method (screen to confirm) that simultaneously achieves high sensitivity and specificity in the diagnosis of MTBC.
| S-EPMC7952592 | biostudies-literature
Project description:Background:The change from non-molecular to nucleic acid amplification tests (NAATs) is known to increase the detection of Clostridium difficile infection (CDI); however, the impact on stool rejection policies in clinical laboratories is unclear. The current guidelines have reinforced the importance of respecting strict conditions for performing tests on stool samples for CDI diagnosis. The purpose of this study was to estimate whether the implementation of molecular tests has resulted in changes in stool rejection policies between clinical laboratories that introduced NAATs and those that did not. Results:A survey was conducted to evaluate the change in the number of stool samples rejected and the rejection criteria among 12 hospital laboratories in southwestern France before and after the switch from non-molecular tests to NAATs using retrospective data from June 1 till September 30, 2013 and the same period 2014. Four laboratories introduced NAATs as a second or third step in the process. A total of 1378 and 1297 stools samples were collected in 2013 and 2014, respectively. The mean number of rejected stool samples significantly increased (p?<?0.001, Chi square test), with a total of 99 (7.1%) and 147 (11.3%) specimens rejected in 2013 and 2014, respectively. Notably, these laboratories had more stringent criteria and were no longer testing the stool samples of patients with CDI-positive results within 7 days. In contrast, there was a significant decrease in the rate of rejected stool samples (p?<?0.001, Chi square test) in the five laboratories that did not adopt NAATs and a less stringent stool rejection policy. Conclusion:Nucleic acid amplification test implementation improved compliance with recommended stool rejection policies. Laboratories should follow the recommended laboratory algorithm for the CDI diagnosis combined with the correct stool rejection policy.
| S-EPMC5975266 | biostudies-literature
Project description:Rapid, automated, point-of-care cellular diagnosis of cancer remains difficult in remote settings due to lack of specialists and medical infrastructure. To address the need for same-day diagnosis, we developed an automated image cytometry system (CytoPAN) that allows rapid breast cancer diagnosis of scant cellular specimens obtained by fine needle aspiration (FNA) of palpable mass lesions. The system is devoid of moving parts for stable operations, harnesses optimized antibody kits for multiplexed analysis, and offers a user-friendly interface with automated analysis for rapid diagnoses. Through extensive optimization and validation using cell lines and mouse models, we established breast cancer diagnosis and receptor subtyping in 1 hour using as few as 50 harvested cells. In a prospective patient cohort study (n = 68), we showed that the diagnostic accuracy was 100% for cancer detection and the receptor subtyping accuracy was 96% for human epidermal growth factor receptor 2 and 93% for hormonal receptors (ER/PR), two key biomarkers associated with breast cancer. A combination of FNA and CytoPAN offers faster, less invasive cancer diagnoses than the current standard (core biopsy and histopathology). This approach should enable the ability to more rapidly diagnose breast cancer in global and remote settings.
| S-EPMC8217912 | biostudies-literature
Project description:Nucleic acid amplification tests (NAATs) provide high diagnostic accuracy for infectious diseases and quantitative results for monitoring viral infections. The majority of NAATs require complex equipment, cold chain dependent reagents, and skilled technicians to perform the tests. This largely confines NAATs to centralized laboratories and can significantly delay appropriate patient care. Low-cost, point-of-care (POC) NAATs are especially needed in low-resource settings to provide patients with diagnosis and treatment planning in a single visit to improve patient care. In this work, we present a rapid POC NAAT with integrated sample preparation and amplification using electrokinetics and paper substrates. We use simultaneous isotachophoresis (ITP) and recombinase polymerase amplification (RPA) to rapidly extract, amplify, and detect target nucleic acids from serum and whole blood in a paper-based format. We demonstrate simultaneous ITP and RPA can consistently detect 5 copies per reaction in buffer and 10 000 copies per milliliter of human serum with no intermediate user steps. We also show preliminary extraction and amplification of DNA from whole blood samples. Our test is rapid (results in less than 20 min) and made from low-cost materials, indicating its potential for detecting infectious diseases and monitoring viral infections at the POC in low resource settings.
| S-EPMC6504979 | biostudies-literature
Project description:The detection of nucleic acid biomarkers for point-of-care (POC) diagnostics is currently limited by technical complexity, cost, and time constraints. To overcome these shortcomings, we have combined loop-mediated isothermal amplification (LAMP), programmable toehold-mediated strand-exchange signal transduction, and standard pregnancy test strips. The incorporation of an engineered hCG-SNAP fusion reporter protein (human chorionic gonadotropin-O6 -alkylguanine-DNA alkyltransferase) led to LAMP-to-hCG signal transduction on low-cost, commercially available pregnancy test strips. Our assay reliably detected as few as 20 copies of Ebola virus templates in both human serum and saliva and could be adapted to distinguish a common melanoma-associated SNP allele (BRAF V600E) from the wild-type sequence. The methods described are completely generalizable to many nucleic acid biomarkers, and could be adapted to provide POC diagnostics for a range of pathogens.
| S-EPMC5240841 | biostudies-literature
Project description:It is inherently difficult to quantitate nucleic acid analytes with most isothermal amplification assays. We developed loop-mediated isothermal amplification (LAMP) reactions in which competition between defined numbers of "false" and "true" amplicons leads to order of magnitude quantitation by a single endpoint determination. These thresholded LAMP reactions were successfully used to directly and quantitatively estimate the numbers of nucleic acids in complex biospecimens, including directly from cells and in sewage, with the values obtained closely correlating with qPCR quantitations. Thresholded LAMP reactions are amenable to endpoint readout by cell phone, unlike other methods that require continuous monitoring, and should therefore prove extremely useful in developing one-pot reactions for point-of-care diagnostics without needing sophisticated material or informatics infrastructure.
| S-EPMC5890436 | biostudies-literature
Project description:Digital nucleic acid amplification tests (digital NAATs) have emerged as a popular tool for nucleic acid detection due to their high sensitivity and specificity. Most current digital NAAT platforms, however, are limited to a "one-color-one-target" approach wherein each target is encoded with a specific fluorescently-labeled probe for single-plex fluorometric detection. This approach is difficult to multiplex due to spectral overlap between any additional fluorophores, and multiplexability of digital NAATs has therefore been limited. As a means to scale multiplexability, we have developed a multiplexed digital NAAT platform, termed Droplet Digital Ratiometric Fluorescence Coding (ddRFC), via a padlock probe-based nucleic acid detection assay which encodes each nucleic acid target with a unique combination of 2 fluorophores. We detect this encoded two-color fluorescence signature of each target by performing digital amplification in microfluidic droplets. To demonstrate the utility of our platform, we have synthesized 6 distinct padlock probes, each rendering a unique two-color fluorescence signature to a nucleic acid target representing a clinically important sexually transmitted infection (STI). We proceed to demonstrate broad-based, two-plex, four-plex, and six-plex detection of the STI targets with single-molecule resolution. Our design offers a cost-effective approach to scale up multiplexability by simply tuning the number of molecular beacon binding sites on the padlock probe without redesigning amplification primers or fluorescent molecular beacons. With further development, our platform has the potential to enable highly multiplexed detection of nucleic acid targets, with potentially unrestricted multiplexability, and serve as a diagnostic tool for many more diseases in the future.
| S-EPMC7534973 | biostudies-literature
Project description:Canine distemper virus (CDV) is a multi-host pathogen that can cause significant mortality in domestic, wild terrestrial and marine mammals. It is a major conservation threat in some endangered species. Infection can result in severe respiratory disease and fatal encephalitis. Diagnosis and disease monitoring in wildlife, and differentiation of CDV from rabies (a life-threatening zoonotic disease that can produce similar neurologic signs), would benefit from the availability of a portable, point-of-care (POC) diagnostic test. We therefore developed a quantitative RT-PCR assay for CDV using shelf-stable, lyophilized reagents and target-specific primers and probes for use with the handheld Biomeme two3™ qPCR thermocycler. Biomeme's extraction methodology, lyophilized reagents, and thermocycler were compared to our standard laboratory-based methods to assess sensitivity, efficiency and overall test performance. Results using a positive control plasmid for CDV showed comparable sensitivity (detection of 50 copies) and PCR efficiency between the two platforms, and CDV detection was similar between platforms when tested using a modified live CDV vaccine. Significantly higher Ct values (average Ct = 5.1 cycles) were observed using the Biomeme platform on known CDV positive animal samples. CDV detection using the Biomeme platform was similar in 25 of 26 samples from suspect CDV cases when compared to standard virology laboratory testing. One false positive was observed that was negative upon retest. The Biomeme methodology can be adapted for detection of specific targets, and this portable technology saves time by eliminating the need for local or international sample transport for laboratory-based diagnostics. However, results of our testing suggest that decreased diagnostic sensitivity (higher Ct values) relative to laboratory-based methods was observed using animal samples, so careful validation and optimization are essential. Portable qPCR platforms can empower biologists and wildlife health professionals in remote and low-resource settings, which will greatly improve our understanding of CDV disease ecology and associated conservation threats in wildlife.
| S-EPMC7176111 | biostudies-literature
Project description:X-ray screening is an important tool in tuberculosis (TB) prevention and care, but access has historically been restricted by its immobile nature. As recent advancements have improved the portability of modern X-ray systems, this study represents an early evaluation of the safety, image quality and yield of using an ultra-portable X-ray system for active case finding (ACF). We reported operational and radiological performance characteristics and compared image quality between the ultra-portable and two reference systems. Image quality was rated by three human readers and by an artificial intelligence (AI) software. We deployed the ultra-portable X-ray alongside the reference system for community-based ACF and described TB care cascades for each system. The ultra-portable system operated within advertised specifications and radiologic tolerances, except on X-ray capture capacity, which was 58% lower than the reported maximum of 100 exposures per charge. The mean image quality rating from radiologists for the ultra-portable system was significantly lower than the reference (3.71 vs. 3.99, p < 0.001). However, we detected no significant differences in TB abnormality scores using the AI software (p = 0.571), nor in any of the steps along the TB care cascade during our ACF campaign. Despite some shortcomings, ultra-portable X-ray systems have significant potential to improve case detection and equitable access to high-quality TB care.
| S-EPMC8482270 | biostudies-literature