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1.
J Food Prot ; 85(1): 44-53, 2022 01 01.
Article in English | MEDLINE | ID: covidwho-1810928

ABSTRACT

ABSTRACT: Vibrio cholerae can cause pandemic cholera in humans. The bacterium resides in aquatic environments worldwide. Continuous testing of V. cholerae contamination in water and aquatic products is imperative for food safety control and human health. In this study, a rapid and visualized method was developed for the first time based on loop-mediated isothermal amplification (LAMP) for detection of the important virulence-related genes ace, zot, cri, and nanH for toxins and the infectious process of V. cholerae. Three pairs of molecular probes targeting each of these genes were designed and synthesized. The one-step LAMP reaction was conducted at 65°C for 40 min. Positive results were inspected by the production of a light green color under visible light or green fluorescence under UV light (302 nm). Limit of detection of the LAMP method ranged from 1.85 to 2.06 pg per reaction of genomic DNA or 2.50 × 100 to 4.00 × 102 CFU per reaction for target genes of cell culture of V. cholerae, which was more sensitive than standard PCR. Inclusivity and exclusivity of the LAMP method were 100% for all target genes. The method showed similar high efficiency to a certain extent in rapid testing of spiked or collected specimens of water and aquatic products. Target genes were detected by absence from all water samples from various sources. However, high occurrences of the nanH gene were observed in intestinal samples derived from four species of fish and one species of shellfish, indicating a risk of potentially toxic V. cholerae in commonly consumed aquatic products. The results in this study provide a potential tool for rapid and visualized detection of V. cholerae in water and aquatic products.


Subject(s)
Vibrio cholerae , Animals , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques/methods , Sensitivity and Specificity , Vibrio cholerae/genetics , Virulence , Water
2.
Front Cell Infect Microbiol ; 11: 778808, 2021.
Article in English | MEDLINE | ID: covidwho-1789350

ABSTRACT

Objectives: Overuse of antibiotics and antibiotic resistance are global healthcare problems. In pediatric patients with respiratory infections, viral and bacterial etiologies are challenging to distinguish, leading to irrational antibiotic use. Rapid and accurate molecular diagnostic testing methods for respiratory pathogens has been shown to facilitate effective clinical decision-making and guide antibiotic stewardship interventions in the developed regions, but its impacts on pediatric patient care in the developing countries remain unclear. Methods: In this single-center, retrospective case-control study, we compared demographics, clinical characteristics, especially microbiological findings, and antibiotic usage between pediatric patients with respiratory infection receiving FilmArray Respiratory Panel (FilmArray RP) testing and a matched routine testing control group. Our primary outcome was the duration of intravenous antibiotics treatment (DOT) during hospitalization. Results: Each group consisted of 346 children with a respiratory infection. In the FilmArray RP testing group, the DOT was shorter than that in the routine testing group (6.41 ± 3.67 days versus 7.23 ± 4.27 days; p = 0.006). More patients in the FilmArray RP testing group de-escalated antibiotic treatments within 72 hours of hospitalization (7.80%, 27/346 versus 2.60%, 9/346; p = 0.002). By contrast, fewer patients in the FilmArray RP testing group had escalated antibiotic treatments between 72 hours and seven days (7.80% versus 14.16%; p = 0.007). The cost of hospitalization was significantly lower in the FilmArray RP testing group ($ 1413.51 ± 1438.01 versus $ 1759.37 ± 1929.22; p = 0.008). Notably, the subgroup analyses revealed that the FilmArray RP test could shorten the DOT, improve early de-escalation of intravenous antibiotics within 72 hours of hospitalization, decline the escalation of intravenous antibiotics between 72 hours and seven days, and reduce the cost of hospitalization for both patient populations with or without underlying diseases. Conclusions: Molecular point-of-care testing for respiratory pathogens could help to reduce intravenous antibiotic use and health care costs of pediatric patients with respiratory infections in developing countries.


Subject(s)
Respiratory Tract Infections , Anti-Bacterial Agents/therapeutic use , Case-Control Studies , Child , Humans , Molecular Diagnostic Techniques , Point-of-Care Testing , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/drug therapy , Retrospective Studies
3.
Front Cell Infect Microbiol ; 12: 799678, 2022.
Article in English | MEDLINE | ID: covidwho-1785317

ABSTRACT

COVID-19 pandemic ignited the development of countless molecular methods for the diagnosis of SARS-CoV-2 based either on nucleic acid, or protein analysis, with the first establishing as the most used for routine diagnosis. The methods trusted for day to day analysis of nucleic acids rely on amplification, in order to enable specific SARS-CoV-2 RNA detection. This review aims to compile the state-of-the-art in the field of nucleic acid amplification tests (NAATs) used for SARS-CoV-2 detection, either at the clinic level, or at the Point-Of-Care (POC), thus focusing on isothermal and non-isothermal amplification-based diagnostics, while looking carefully at the concerning virology aspects, steps and instruments a test can involve. Following a theme contextualization in introduction, topics about fundamental knowledge on underlying virology aspects, collection and processing of clinical samples pave the way for a detailed assessment of the amplification and detection technologies. In order to address such themes, nucleic acid amplification methods, the different types of molecular reactions used for DNA detection, as well as the instruments requested for executing such routes of analysis are discussed in the subsequent sections. The benchmark of paradigmatic commercial tests further contributes toward discussion, building on technical aspects addressed in the previous sections and other additional information supplied in that part. The last lines are reserved for looking ahead to the future of NAATs and its importance in tackling this pandemic and other identical upcoming challenges.


Subject(s)
COVID-19 , Nucleic Acids , COVID-19/diagnosis , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
4.
PLoS One ; 17(4): e0266703, 2022.
Article in English | MEDLINE | ID: covidwho-1785202

ABSTRACT

In 2019 a newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly from the epicenter in Wuhan (China) to more than 150 countries around the world, causing the Coronavirus disease 2019 (COVID-19) pandemic. In this study, we describe an extraction-less method based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) intended for the rapid qualitative detection of nucleic acid from SARS-CoV-2 in upper respiratory specimens, including oropharyngeal and nasopharyngeal swabs, anterior nasal and mid-turbinate nasal swabs, nasopharyngeal washes/aspirates or nasal aspirates as well as bronchoalveolar lavage (BAL) from individuals suspected of COVID-19 by their healthcare provider. The assay's performance was evaluated and compared to an RT quantitative PCR-based assay (FDA-approved). With high sensitivity, specificity, and bypassing the need for RNA extraction, the RT-LAMP Rapid Detection assay is a valuable and fast test for an accurate and rapid RNA detection of the SARS-CoV-2 virus and potentially other pathogens. Additionally, the versatility of this test allows its application in virtually every laboratory setting and remote location where access to expensive laboratory equipment is a limiting factor for testing during pandemic crises.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Cost-Benefit Analysis , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
5.
Sci Rep ; 12(1): 5936, 2022 Apr 08.
Article in English | MEDLINE | ID: covidwho-1784018

ABSTRACT

Without any realistic prospect of comprehensive global vaccine coverage and lasting immunity, control of pandemics such as COVID-19 will require implementation of large-scale, rapid identification and isolation of infectious individuals to limit further transmission. Here, we describe an automated, high-throughput integrated screening platform, incorporating saliva-based loop-mediated isothermal amplification (LAMP) technology, that is designed for population-scale sensitive detection of infectious carriers of SARS-CoV-2 RNA. Central to this surveillance system is the "Sentinel" testing instrument, which is capable of reporting results within 25 min of saliva sample collection with a throughput of up to 3840 results per hour. It incorporates continuous flow loading of samples at random intervals to cost-effectively adjust for fluctuations in testing demand. Independent validation of our saliva-based RT-LAMP technology on an automated LAMP instrument coined the "Sentinel", found 98.7% sensitivity, 97.6% specificity, and 98% accuracy against a RT-PCR comparator assay, confirming its suitability for surveillance screening. This Sentinel surveillance system offers a feasible and scalable approach to complement vaccination, to curb the spread of COVID-19 variants, and control future pandemics to save lives.


Subject(s)
COVID-19 , Saliva , COVID-19/diagnosis , COVID-19/epidemiology , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Saliva/chemistry , Sensitivity and Specificity
6.
Sci Rep ; 12(1): 5729, 2022 Apr 06.
Article in English | MEDLINE | ID: covidwho-1778632

ABSTRACT

The Coronavirus disease 2019 (COVID-19) pandemic-caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)- has posed a global threat and presented with it a multitude of economic and public-health challenges. Establishing a reliable means of readily available, rapid diagnostic testing is of paramount importance in halting the spread of COVID-19, as governments continue to ease lockdown restrictions. The current standard for laboratory testing utilizes reverse transcription quantitative polymerase chain reaction (RT-qPCR); however, this method presents clear limitations in requiring a longer run-time as well as reduced on-site testing capability. Therefore, we investigated the feasibility of a reverse transcription looped-mediated isothermal amplification (RT-LAMP)-based model of rapid COVID-19 diagnostic testing which allows for less invasive sample collection, named SaliVISION. This novel, two-step, RT-LAMP assay utilizes a customized multiplex primer set specifically targeting SARS-CoV-2 and a visual report system that is ready to interpret within 40 min from the start of sample processing and does not require a BSL-2 level testing environment or special laboratory equipment. When compared to the SalivaDirect and Thermo Fisher Scientific TaqPath RT-qPCR testing platforms, the respective sensitivities of the SaliVISION assay are 94.29% and 98.28% while assay specificity was 100% when compared to either testing platform. Our data illustrate a robust, rapid diagnostic assay in our novel RT-LAMP test design, with potential for greater testing throughput than is currently available through laboratory testing and increased on-site testing capability.


Subject(s)
COVID-19 , COVID-19/diagnosis , COVID-19 Testing , Communicable Disease Control , Diagnostic Tests, Routine , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Saliva/chemistry , Sensitivity and Specificity
7.
Mikrochim Acta ; 189(5): 176, 2022 Apr 05.
Article in English | MEDLINE | ID: covidwho-1777733

ABSTRACT

A probing system has been developed based on dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for the selective colorimetric detection of SARS-CoV-2. This approach can induce false-positive and -negative detection in real clinical samples; dLig-LAMP operates with improved selectivity. Unlike RT-LAMP, the selectivity of dLig-LAMP is determined in both the ligation and primer binding steps, not in the reverse transcription step. With this selective system in hand, we developed a colorimetric signaling system for point-of-care detection. We also developed a colorimetric probe for sensing pyrophosphate, which arises as a side product during the LAMP DNA amplification. Thus, dLig-LAMP appears to be an alternative method for improving the selectivity problems associated with reverse transcription. In addition, combining dLig-LAMP with colorimetric pyrophosphate probing allows point-of-care detection of SARS-CoV-2 within 1 h with high selectivity.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Colorimetry/methods , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques/methods , Point-of-Care Systems , SARS-CoV-2/genetics
8.
J Med Virol ; 94(5): 1998-2007, 2022 05.
Article in English | MEDLINE | ID: covidwho-1777578

ABSTRACT

Coronavirus disease 2019 or COVID-19 caused by novel coronavirus/severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or 2019-nCoV) is an ongoing pandemic that has emerging global effects and requires rapid and reliable diagnostic testing. Quantitative reverse transcription-polymerase chain reaction (q-RT-PCR) is the gold standard method for SARS-CoV-2 detections. On the other hand, new approaches remedy the diagnosis difficulties gradually. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) as one of these novel approaches may also contribute to faster and cheaper field-based testing. The present study was designed to evaluate this rapid screening diagnostic test that can give results in 30-45 min and to compare the effectiveness of LAMP to the q-RT-PCR. The 30 randomly chosen patient samples were generated by nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. The sample of quantification cycle (Cq) values was tested using RT-LAMP as well as by conventional q-RT-PCR. The patient samples were tested with four different kits (SENSObiz COVID-19 [SARS-CoV-2] LAMP Assay, the QIAseq DIRECT SARS-CoV-2 kit, Biospeedy SARS-CoV-2 Variant Plus kit, and CoVirion-CV19-2 SARS-CoV-2 OneStep RT-PCR kit) and two different PCR devices (GDS Rotor-Gene Q Thermocycler and Inovia Technologies GenX series). Based on 30 patient samples, the positive/negative ratio (P/N) was 30/0 as Biospeedy and Covirion (positivity 100%), 28/2 as Qiagen kit (positivity 93.3%) for the samples studied on the Inovia device while the same samples on the Rotor-Gene device were 30/0 as Biospeedy and Covirion (positivity 100%), 29/1 as Qiagen kit at the first day (96.7%). On the fifth day, the samples were studied in the Inovia device and the respective results were obtained: 27/3 as Biospeedy (positivity 90%), 16/14 as Qiagen (positivity 53.3%), 28/2 as Covirion kit (positivity 93.3%). When these samples were studied in the Rotor-Gene device, it was 29/1 in Biospeedy and Covirion (positivity 96.7%), 19/11 in the Qiagen kit (positivity 63.3%). When these samples were compared with the LAMP method it was found to be 19/11 (positivity 63.3%) on the first day and 18/12 (positivity 60%) on the fifth day. SARS-CoV-2 test studies will contribute to a proactive approach to the development of rapid diagnosis systems. The LAMP approach presents promising results to monitor exposed individuals and also improves screening efforts in potential ports of entry.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Clinical Laboratory Techniques/methods , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity
9.
Commun Biol ; 5(1): 290, 2022 Mar 31.
Article in English | MEDLINE | ID: covidwho-1774000

ABSTRACT

Nucleic acid detection is essential for numerous biomedical applications, but often requires complex protocols and/or suffers false-positive readouts. Here, we describe SENTINEL, an approach that combines isothermal amplification with a sequence-specific degradation method to detect nucleic acids with high sensitivity and sequence-specificity. Target single-stranded RNA or double-stranded DNA molecules are amplified by loop-mediated isothermal amplification (LAMP) and subsequently degraded by the combined action of lambda exonuclease and a sequence-specific DNA endonuclease (e.g., Cas9). By combining the sensitivity of LAMP with the precision of DNA endonucleases, the protocol achieves attomolar limits of detection while differentiating between sequences that differ by only one or two base pairs. The protocol requires less than an hour to complete using a 65 °C heat block and fluorometer, and detects SARS-CoV-2 virus particles in human saliva and nasopharyngeal swabs with high sensitivity.


Subject(s)
COVID-19 , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Nucleic Acids , COVID-19/diagnosis , DNA , Endonucleases , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Nucleic Acids/isolation & purification , SARS-CoV-2/genetics
10.
PLoS One ; 17(4): e0254324, 2022.
Article in English | MEDLINE | ID: covidwho-1770645

ABSTRACT

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has emerged as a viable molecular diagnostic method to expand the breadth and reach of nucleic acid testing, particularly for SARS-CoV-2 detection and surveillance. While rapidly growing in prominence, RT-LAMP remains a relatively new method compared to the standard RT-qPCR, and contribution to our body of knowledge on designing LAMP primer sets and assays can have significant impact on its utility and adoption. Here we select and evaluate 18 LAMP primer sets for SARS-CoV-2 previously identified as sensitive ones under various conditions, comparing their speed and sensitivity with two LAMP formulations each with 2 reaction temperatures. We find that both LAMP formulations have some effects on the speed and detection sensitivity and identify several primer sets with similar high sensitivity for different SARS-CoV-2 gene targets. Significantly we observe a consistent sensitivity enhancement by combining primer sets for different targets, confirming and building on earlier work to create a simple, general approach to building better and more sensitive RT-LAMP assays.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
11.
Biosensors (Basel) ; 12(3)2022 Mar 02.
Article in English | MEDLINE | ID: covidwho-1760369

ABSTRACT

Campylobacter jejuni is one of the most important causes of food-borne infectious disease, and poses challenges to food safety and public health. Establishing a rapid, accurate, sensitive, and simple detection method for C. jejuni enables early diagnosis, early intervention, and prevention of pathogen transmission. In this study, an immunocapture magnetic bead (ICB)-enhanced loop-mediated isothermal amplification (LAMP) CRISPR/Cas12a method (ICB-LAMP-CRISPR/Cas12a) was developed for the rapid and visual detection of C. jejuni. Using the ICB-LAMP-CRISPR/Cas12a method, C. jejuni was first captured by ICB, and the bacterial genomic DNA was then released by heating and used in the LAMP reaction. After the LAMP reaction, LAMP products were mixed and detected by the CRISPR/Cas12a cleavage mixture. This ICB-LAMP-CRISPR/Cas12a method could detect a minimum of 8 CFU/mL of C. jejuni within 70 min. Additionally, the method was performed in a closed tube in addition to ICB capture, which eliminates the need to separate preamplification and transfer of amplified products to avoid aerosol pollution. The ICB-LAMP-CRISPR/Cas12a method was further validated by testing 31 C. jejuni-positive fecal samples from different layer farms. This method is an all-in-one, simple, rapid, ultrasensitive, ultraspecific, visual detection method for instrument-free diagnosis of C. jejuni, and has wide application potential in future work.


Subject(s)
Campylobacter jejuni , CRISPR-Cas Systems , Magnetic Phenomena , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques/methods
12.
PLoS One ; 17(3): e0259610, 2022.
Article in English | MEDLINE | ID: covidwho-1759938

ABSTRACT

The ongoing SARS-CoV-2 pandemic has necessitated a dramatic increase in our ability to conduct molecular diagnostic tests, as accurate detection of the virus is critical in preventing its spread. However, SARS-CoV-2 variants continue to emerge, with each new variant potentially affecting widely-used nucleic acid amplification diagnostic tests. RT-LAMP has been adopted as a quick, inexpensive diagnostic alternative to RT-qPCR, but as a newer method, has not been studied as thoroughly. Here we interrogate the effect of SARS-CoV-2 sequence mutations on RT-LAMP amplification, creating 523 single point mutation "variants" covering every position of the LAMP primers in 3 SARS-CoV-2 assays and analyzing their effects with over 4,500 RT-LAMP reactions. Remarkably, we observed only minimal effects on amplification speed and no effect on detection sensitivity at positions equivalent to those that significantly impact RT-qPCR assays. We also created primer sets targeting a specific short deletion and observed that LAMP is able to amplify even with a primer containing multiple consecutive mismatched bases, albeit with reduced speed and sensitivity. This highlights RT-LAMP as a robust technique for viral RNA detection that can tolerate most mutations in the primer regions. Additionally, where variant discrimination is desired, we describe the use of molecular beacons to sensitively distinguish and identify variant RNA sequences carrying short deletions. Together these data add to the growing body of knowledge on the utility of RT-LAMP and increase its potential to further our ability to conduct molecular diagnostic tests outside of the traditional clinical laboratory environment.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
13.
Sci Rep ; 12(1): 4947, 2022 03 23.
Article in English | MEDLINE | ID: covidwho-1758377

ABSTRACT

The COVID-19 pandemic required increased testing capacity, enabling rapid case identification and effective contract tracing to reduce transmission of disease. The BioFire FilmArray is a fully automated nucleic acid amplification test system providing specificity and sensitivity associated with gold standard molecular methods. The FilmArray Respiratory Panel 2.1 targets 22 viral and bacterial pathogens, including SARS-CoV-2 and influenza virus. While each panel provides a robust output of information regarding pathogen detection, the specimen throughput is low. This study evaluates the FilmArray Respiratory Panel 2.1 using 33 pools of contrived nasal samples and 22 pools of clinical nasopharyngeal specimens to determine the feasibility of increasing testing capacity, while maintaining detection of both SARS-CoV-2 and influenza virus. We observed 100% detection and 90% positive agreement for SARS-CoV-2 and 98% detection and 95% positive agreement for influenza viruses with pools of contrived or clinical specimens, respectively. While discordant results were mainly attributed to loss in sensitivity, the sensitivity of the pooling assay was well within accepted limits of detection for a nucleic acid amplification test. Overall, this study provides evidence supporting the use of pooling patient specimens, one in four with the FilmArray Respiratory Panel 2.1 for the detection of SARS-CoV-2 and influenza virus.


Subject(s)
COVID-19 , Orthomyxoviridae , Respiratory Tract Infections , COVID-19/diagnosis , Humans , Molecular Diagnostic Techniques/methods , Orthomyxoviridae/genetics , Pandemics , SARS-CoV-2/genetics
14.
PLoS One ; 17(3): e0265748, 2022.
Article in English | MEDLINE | ID: covidwho-1753205

ABSTRACT

The new coronavirus infection (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be fatal, and several variants of SARS-CoV-2 with mutations of the receptor-binding domain (RBD) have increased avidity for human cell receptors. A single missense mutation of U to G at nucleotide position 1355 (U1355G) in the spike (S) gene changes leucine to arginine (L452R) in the spike protein. This mutation has been observed in the India and California strains (B.1.617 and B.1.427/B.1.429, respectively). Control of COVID-19 requires rapid and reliable detection of SARS-CoV-2. Therefore, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay plus a bioluminescent assay in real-time (BART) to detect SARS-CoV-2 and the L452R spike mutation. The specificity and sensitivity of the RT-LAMP-BART assay was evaluated using synthetic RNAs including target sequences and RNA-spiked clinical nasopharyngeal and saliva specimens as well as reference strains representing five viral and four bacterial pathogens. The novel RT-LAMP-BART assay to detect SARS-CoV-2 was highly specific compared to the conventional real-time RT-PCR. Within 25 min, the RT-LAMP-BART assay detected 80 copies of the target gene in a sample, whereas the conventional real-time RT-PCR method detected 5 copies per reaction within 130 min. Using RNA-spiked specimens, the sensitivity of the RT-LAMP-BART assay was slightly attenuated compared to purified RNA as a template. The results were identical to those of the conventional real-time RT-PCR method. Furthermore, using a peptide nucleic acid (PNA) probe, the RT-LAMP-BART method correctly identified the L452R spike mutation. This is the first report describes RT-LAMP-BART as a simple, inexpensive, rapid, and useful assay for detection of SARS-CoV-2, its variants of concern, and for screening of COVID-19.


Subject(s)
Amino Acid Substitution , COVID-19/diagnosis , Peptide Nucleic Acids/genetics , SARS-CoV-2/classification , Spike Glycoprotein, Coronavirus/genetics , Binding Sites , California , Early Diagnosis , Humans , India , Limit of Detection , Luminescent Measurements , Molecular Diagnostic Techniques , Mutation, Missense , Nucleic Acid Amplification Techniques , Real-Time Polymerase Chain Reaction , Reverse Transcription , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/chemistry
15.
Emerg Microbes Infect ; 11(1): 978-987, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1740711

ABSTRACT

The highly infectious Delta variant strain of SARS-CoV-2 remains globally dominant and undermines COVID-19 vaccines. Rapid detection of the Delta variant is crucial for the identification and quarantine of infected individuals. In this study, our aim was to design and validate a genotyping RT-LAMP method to detect Delta variants specifically. R203M in the N gene of SARS-CoV-2 was chosen as the Delta variant-specific mutation for genotyping. To target the R203M-harboring region and the conserved sequence of the N gene, two sets of primers were designed, and a Cq (quantification cycle) ratio-based RT-LAMP for SARS-CoV-2 and R203M detection was developed by analyzing the significant discrepancy in amplification efficiency of the two sets of primers. We validated the RT-LAMP method on 498 clinical specimens in parallel with RT-qPCR, and 84 Delta variants from 198 positive samples were determined by sequencing. Compared with traditional RT-qPCR analyses, RT-LAMP appears to be 100% accurate in detecting SARS-CoV-2 clinical samples. RT-LAMP has a good ability to distinguish between Delta and non-Delta variants under a Cq ratio threshold of 1.80. Furthermore, the AUC (area under the curve) of this method was 1.00; the sensitivity, specificity and accuracy were all 100%. In summary, we have proposed a rapid, accurate and cost-effective RT-LAMP method to detect SARS-CoV-2 and Delta variants, which may facilitate the surveillance of COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Vaccines , Humans , Molecular Diagnostic Techniques , Mutation , Nucleic Acid Amplification Techniques , SARS-CoV-2/genetics , Sensitivity and Specificity
16.
Sci Rep ; 12(1): 3951, 2022 03 10.
Article in English | MEDLINE | ID: covidwho-1740471

ABSTRACT

The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , SARS-CoV-2/genetics , Saliva/virology , COVID-19 Nucleic Acid Testing/instrumentation , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Real-Time Polymerase Chain Reaction , Specimen Handling/instrumentation , Specimen Handling/methods
17.
Sci Rep ; 12(1): 4132, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735286

ABSTRACT

This paper presents a deep learning-driven portable, accurate, low-cost, and easy-to-use device to perform Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) to facilitate rapid detection of COVID-19. The 3D-printed device-powered using only a 5 Volt AC-DC adapter-can perform 16 simultaneous RT-LAMP reactions and can be used multiple times. Moreover, the experimental protocol is devised to obviate the need for separate, expensive equipment for RNA extraction in addition to eliminating sample evaporation. The entire process from sample preparation to the qualitative assessment of the LAMP amplification takes only 45 min (10 min for pre-heating and 35 min for RT-LAMP reactions). The completion of the amplification reaction yields a fuchsia color for the negative samples and either a yellow or orange color for the positive samples, based on a pH indicator dye. The device is coupled with a novel deep learning system that automatically analyzes the amplification results and pays attention to the pH indicator dye to screen the COVID-19 subjects. The proposed device has been rigorously tested on 250 RT-LAMP clinical samples, where it achieved an overall specificity and sensitivity of 0.9666 and 0.9722, respectively with a recall of 0.9892 for Ct < 30. Also, the proposed system can be widely used as an accurate, sensitive, rapid, and portable tool to detect COVID-19 in settings where access to a lab is difficult, or the results are urgently required.


Subject(s)
COVID-19/diagnosis , Deep Learning , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , Area Under Curve , COVID-19 Testing , Coloring Agents/chemistry , Humans , Molecular Diagnostic Techniques/instrumentation , Nasopharynx/virology , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Systems , Printing, Three-Dimensional , RNA, Viral/analysis , RNA, Viral/metabolism , ROC Curve , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
18.
Sci Rep ; 12(1): 3775, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735272

ABSTRACT

Loop-mediated isothermal amplification is known for its high sensitivity, specificity and tolerance to inhibiting-substances. In this work, we developed a device for performing real-time colorimetric LAMP combining the accuracy of lab-based quantitative analysis with the simplicity of point-of-care testing. The device innovation lies on the use of a plastic tube anchored vertically on a hot surface while the side walls are exposed to a mini camera able to take snapshots of the colour change in real time during LAMP amplification. Competitive features are the rapid analysis (< 30 min), quantification over 9 log-units, crude sample-compatibility (saliva, tissue, swabs), low detection limit (< 5 copies/reaction), smartphone-operation, fast prototyping (3D-printing) and ability to select the dye of interest (Phenol red, HNB). The device's clinical utility is demonstrated in cancer mutations-analysis during the detection of 0.01% of BRAF-V600E-to-wild-type molecules from tissue samples and COVID-19 testing with 97% (Ct < 36.8) and 98% (Ct < 30) sensitivity when using extracted RNA and nasopharyngeal-swabs, respectively. The device high technology-readiness-level makes it a suitable platform for performing any colorimetric LAMP assay; moreover, its simple and inexpensive fabrication holds promise for fast deployment and application in global diagnostics.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , Colorimetry , Humans , Limit of Detection , Molecular Diagnostic Techniques , Nasopharynx/virology , Neoplasms/diagnosis , Neoplasms/genetics , Neoplasms/pathology , Nucleic Acid Amplification Techniques , Point-of-Care Testing , Proto-Oncogene Proteins B-raf/genetics , RNA, Viral/analysis , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Smartphone
19.
Lab Chip ; 22(7): 1297-1309, 2022 03 29.
Article in English | MEDLINE | ID: covidwho-1730327

ABSTRACT

Since the beginning of the COVID-19 pandemic, several mutations of the SARS-CoV-2 virus have emerged. Current gold standard detection methods for detecting the virus and its variants are based on PCR-based diagnostics using complex laboratory protocols and time-consuming steps, such as RNA isolation and purification, and thermal cycling. These steps limit the translation of technology to the point-of-care and limit accessibility to under-resourced regions. While PCR-based assays currently offer the possibility of multiplexed gene detection, and commercial products of single gene PCR and isothermal LAMP at point-of-care are also now available, reports of isothermal assays at the point-of-care with detection of multiple genes are lacking. Here, we present a microfluidic assay and device to detect and differentiate the Alpha variant (B.1.1.7) from the SARS-CoV-2 virus early strains in saliva samples. The detection assay, which is based on isothermal RT-LAMP amplification, takes advantage of the S-gene target failure (SGTF) to differentiate the Alpha variant from the SARS-CoV-2 virus early strains using a binary detection system based on spatial separation of the primers specific to the N- and S-genes. We use additively manufactured plastic cartridges in a low-cost optical reader system to successfully detect the SARS-CoV-2 virus from saliva samples (positive amplification is detected with concentration ≥10 copies per µL) within 30 min. We demonstrate that our platform can discriminate the B.1.1.7 variant (USA/CA_CDC_5574/2020 isolate) from SARS-CoV-2 negative samples, but also from the SARS-CoV-2 USA-WA1/2020 isolate. The reliability of the developed point-of-care device was confirmed by testing 38 clinical saliva samples, including 20 samples positive for Alpha variant (sensitivity > 90%, specificity = 100%). This study highlights the current relevance of binary-based testing, as the new Omicron variant also exhibits S-gene target failure and could be tested by adapting the approach presented here.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Microfluidics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , Point-of-Care Systems , RNA, Viral/analysis , RNA, Viral/genetics , Reproducibility of Results , SARS-CoV-2/genetics , Sensitivity and Specificity
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