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1.
PLoS One ; 19(6): e0304497, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38870181

RESUMO

Tomato mosaic virus (ToMV), an economically important virus that affects a wide range of crops, is highly contagious, and its transmission is mediated by mechanical means, and through contaminated seeds or planting materials, making its management challenging. To contain its wide distribution, early and accurate detection of infection is required. A survey was conducted between January and May, 2023 in major tomato growing counties in Kenya, namely, Baringo, Kajiado, Kirinyaga and Laikipia, to establish ToMV disease incidence and to collect samples for optimization of the reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) assay. A RT-LAMP assay, utilizing primers targeting the coat protein, was developed and evaluated for its performance. The method was able to detect ToMV in tomato samples within 4:45 minutes, had a 1,000-fold higher sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR) method and was specific to ToMV. Furthermore, the practical applicability of the assay was assessed using tomato samples and other solanaecous plants. The assay was able to detect the virus in 14 tomato leaf samples collected from the field, compared to 11 samples detected by RT-PCR, further supporting the greater sensitivity of the assay. To make the assay more amenable for on-site ToMV detection, a quick-extraction method based on alkaline polyethylene glycol buffer was evaluated, which permitted the direct detection of the target virus from crude leaf extracts. Due to its high sensitivity, specificity and rapidity, the RT-LAMP method could be valuable for field surveys and quarantine inspections towards a robust management of ToMV infections.


Assuntos
Técnicas de Amplificação de Ácido Nucleico , Doenças das Plantas , Solanum lycopersicum , Tobamovirus , Técnicas de Amplificação de Ácido Nucleico/métodos , Solanum lycopersicum/virologia , Doenças das Plantas/virologia , Tobamovirus/genética , Tobamovirus/isolamento & purificação , Transcrição Reversa , Sensibilidade e Especificidade , Quênia , RNA Viral/genética , RNA Viral/análise , RNA Viral/isolamento & purificação , Técnicas de Diagnóstico Molecular
2.
Anal Chim Acta ; 1314: 342799, 2024 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-38876521

RESUMO

BACKGROUND: As a core enzyme in the base excision repair system, uracil DNA glycosylase (UDG) is indispensable in maintaining genomic integrity and normal cell cycles. Its abnormal activity intervenes in cancers and neurodegerative diseases. Previous UDG assays based on isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) system were fine in sensitivity, but exposed to complications in assay flow, time, and probe design. After isothermal amplification, a CRISPR/Cas reagent should be separately added with extra manual steps and its guide RNA (gRNA) should be designed, considering the presence of protospacer adjacent motif (PAM) site. RESULTS: We herein describe a UDG-REtarded CRISPR Amplification assay, termed 'URECA'. In URECA, isothermal nucleic acid (NA) amplification and CRISPR/Cas12a system were tightly combined to constitute a one-pot, isothermal CRISPR amplification system. Isothermal NA amplification for a UDG substrate (US) with uracil (U) bases was designed to activate and boost CRISPR/Cas12a reaction. Such scheme enabled us to envision that UDG would halt the isothermal CRISPR amplification reaction by excising U bases and messing up the US. Based on this principle, the assay detected the UDG activity down to 9.17 x 10-4 U/mL in 50 min. With URECA, we fulfilled the recovery test of UDG activities in plasma and urine with high precision and reproducibility and reliably determined UDG activities in cell extracts. Also, we verified its capability to screen candidate UDG inhibitors, showing its potentials in practical application as well as drug discovery. SIGNIFICANCE: URECA offers further merits: i) the assay is seamless. Following target recognition, the reactions proceed in one-step without any intervening steps, ii) probe design is simple. Unlike the conventional CRISPR/Cas12a-based assays, URECA does not consider the PAM site in probe design as Cas12a activation relies on instantaneous gRNA binding to single-stranded DNA strands. By rationally designing an enzyme substrate probe to be specific to other enzymes, while keeping a role as a template for isothermal CRISPR amplification, the detection principle of URECA will be expanded to devise biosensors for various enzymes of biological, clinical significance.


Assuntos
Sistemas CRISPR-Cas , Reparo do DNA , Técnicas de Amplificação de Ácido Nucleico , Uracila-DNA Glicosidase , Uracila-DNA Glicosidase/metabolismo , Uracila-DNA Glicosidase/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas CRISPR-Cas/genética , Ensaios Enzimáticos/métodos , Reparo por Excisão
3.
Sci Rep ; 14(1): 13782, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38877073

RESUMO

Kaposi's sarcoma (KS) is a cancer affecting skin and internal organs for which the Kaposi's sarcoma associated herpesvirus (KSHV) is a necessary cause. Previous work has pursued KS diagnosis by quantifying KSHV DNA in skin biopsies using a point-of-care (POC) device which performs quantitative loop-mediated isothermal amplification (LAMP). These previous studies revealed that extracting DNA from patient biopsies was the rate limiting step in an otherwise rapid process. In this study, a simplified, POC-compatible alkaline DNA extraction, ColdSHOT, was optimized for 0.75 mm human skin punch biopsies. The optimized ColdSHOT extraction consistently produced 40,000+ copies of DNA per 5 µl reaction from 3 mg samples-a yield comparable to standard spin column extractions-within 1 h without significant equipment. The DNA yield was estimated sufficient for KSHV detection from KS-positive patient biopsies, and the LAMP assay was not affected by non-target tissue in the unpurified samples. Furthermore, the yields achieved via ColdSHOT were robust to sample storage in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer prior to DNA extraction, and the DNA sample was stable after extraction. The results presented in this study indicate that the ColdSHOT DNA extraction could be implemented to simplify and accelerate the LAMP-based diagnosis of Kaposi's sarcoma using submillimeter biopsy samples.


Assuntos
DNA Viral , Herpesvirus Humano 8 , Técnicas de Amplificação de Ácido Nucleico , Sarcoma de Kaposi , Pele , Humanos , DNA Viral/genética , DNA Viral/isolamento & purificação , Herpesvirus Humano 8/isolamento & purificação , Herpesvirus Humano 8/genética , Biópsia/métodos , Pele/virologia , Pele/patologia , Sarcoma de Kaposi/diagnóstico , Sarcoma de Kaposi/virologia , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Técnicas de Diagnóstico Molecular/métodos
4.
Sci Rep ; 14(1): 13739, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38877111

RESUMO

The study aimed to develop a quantitative colorimetric loop-mediated isothermal amplification technique using the phenol red indicator (QLAMP-PhR) for detecting Fusobacterium nucleatum (Fn) levels in colorectal cancer (CRC) patients and healthy individuals. QLAMP-PhR assays were conducted on 251 stool samples specific for the Fn FadA gene. Six primers were synthesized and utilized with master mix reagents, and a phenol red indicator was employed to enhance the QLAMP-PhR technique. A standard quantitative analysis curve was generated using a logarithmic function (absorbance vs. concentration) by serially diluting the copy number of genomic DNA templates (Fn ATCC25586). The CRC group exhibited a significantly higher abundance of Fn compared to the healthy control group (P < 0.001). These findings suggest that the QLAMP-PhR technique effectively identifies Fn specifically by its gene for the key virulence factor FadA. Additionally, ideas for developing a real-time QLAMP-PhR test were presented. Compared to the traditional polymerase chain reaction (PCR) technique, QLAMP-PhR offers several advantages including rapidity, simplicity, specificity, sensitivity, and cost-effectiveness method that can quantitatively screen for Fn presence in normal populations. The QLAMP-PhR method represents a sensitive and specific amplification assay for the rapid detection of the Fn pathogen. To the best of our knowledge, this study is the first to report the application of QLAMP-PhR for detecting FadA in Fn.


Assuntos
Neoplasias Colorretais , Colorimetria , Fezes , Fusobacterium nucleatum , Técnicas de Amplificação de Ácido Nucleico , Humanos , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Fusobacterium nucleatum/genética , Fusobacterium nucleatum/isolamento & purificação , Fezes/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Colorimetria/métodos , Masculino , Feminino , Fenolsulfonaftaleína , Técnicas de Diagnóstico Molecular/métodos , Pessoa de Meia-Idade , Idoso , Infecções por Fusobacterium/microbiologia , Infecções por Fusobacterium/diagnóstico , Sensibilidade e Especificidade , Adulto
5.
Mikrochim Acta ; 191(7): 397, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38877314

RESUMO

A fluorescence biosensor for determination of aflatoxin B1 (AFB1) based on polydiacetylene (PDA) liposomes and exonuclease III (EXO III)-assisted recycling amplification was developed. The AFB1 aptamer partially hybridizes with complementary DNA (cDNA), which is released upon recognition of AFB1 by the aptamer. Subsequently, the cDNA hybridizes with hairpin H to form double-stranded DNA that undergoes digestion by EXO III, resulting in the cyclic release of cDNA and generation of capture DNA for further reaction. The capture DNA then hybridizes with probe modified on PDA liposomes, leading to aggregation of liposomes and subsequent fluorescence production. This strategy exhibited a limit of detection of 0.18 ng/mL within the linear range 1-100 ng/mL with a determination coefficient > 0.99. The recovery ranged from 92.81 to 106.45%, with relative standard deviations (RSD) between 1.73 and 4.26%, for corn, brown rice, peanut butter, and wheat samples. The stability, accuracy, and specificity of the method demonstrated the applicability for real sample analysis.


Assuntos
Aflatoxina B1 , Técnicas Biossensoriais , Exodesoxirribonucleases , Limite de Detecção , Lipossomos , Polímero Poliacetilênico , Polímero Poliacetilênico/química , Lipossomos/química , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Técnicas Biossensoriais/métodos , Aflatoxina B1/análise , Aptâmeros de Nucleotídeos/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Poli-Inos/química , Espectrometria de Fluorescência/métodos , Zea mays/química , Triticum/química , Oryza/química , Polímeros/química , Contaminação de Alimentos/análise
6.
Genome Biol ; 25(1): 157, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38877540

RESUMO

Methylation-based liquid biopsies show promises in detecting cancer using circulating cell-free DNA; however, current limitations impede clinical application. Most assays necessitate substantial DNA inputs, posing challenges. Additionally, underrepresented tumor DNA fragments may go undetected during exponential amplification steps of traditional sequencing methods. Here, we report linear amplification-based bisulfite sequencing (LABS), enabling linear amplification of bisulfite-treated DNA fragments in a genome-wide, unbiased fashion, detecting cancer abnormalities with sub-nanogram inputs. Applying LABS to 100 patient samples revealed cancer-specific patterns, copy number alterations, and enhanced cancer detection accuracy by identifying tissue-of-origin and immune cell composition.


Assuntos
Metilação de DNA , Neoplasias , Análise de Sequência de DNA , Sulfitos , Humanos , Neoplasias/genética , Análise de Sequência de DNA/métodos , Ácidos Nucleicos Livres , Técnicas de Amplificação de Ácido Nucleico/métodos , Variações do Número de Cópias de DNA , DNA de Neoplasias/genética , DNA Tumoral Circulante/genética
7.
Anal Chim Acta ; 1315: 342797, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38879209

RESUMO

BACKGROUND: Harmful algal blooms (HABs), caused by the rapid proliferation or aggregation of microorganisms, are catastrophic for the environment. The Prymnesium parvum is a haptophyte algal species that is found worldwide and is responsible for extensive blooms and death of larval amphibians and bivalves, causing serious negative impacts on the ecological environment. For the prevention and management of environmental pollution, it is crucial to explore and develop early detection strategies for HABs on-site using simple methods. The major challenge related to early detection is the accurate and sensitive detection of algae present in low abundance. RESULTS: Herein, recombinase polymerase amplification (RPA) was combined with clustered regularly interspaced short palindromic repeats and Cas12a protein (CRISPR-LbaCas12a) systems, and the lateral flow dipstick (LFD) was used for the first time for early detection of P. parvum. The internal transcribed spacer (ITS) of P. parvum was selected as the target sequence, and the concentration of single-strand DNA reporters, buffer liquid system, reaction time, and amount of gold particles were optimized. The RPA-CRISPR-LbaCas12a-LFD approach demonstrated highly specificity during experimental testing, with no cross-reaction against different microalgae used as controls. In addition, the lowest detection limit was 10,000 times better than the lowest detection limit of the standalone RPA approach. The feasibility and robustness of this approach were further verified by using the different environmental samples. It also observed that P. parvum are widely distributed in Chinese Sea, but the cell density of P. parvum is relatively low (<0.1 cells/mL). SIGNIFICANCE: The developed approach has an excellent specificity and offers 10,000 times better sensitivity than the standalone RPA approach. These advantages make this approach suitable for early warning detection and prevention of HAB events in environmental water. Also, the outcomes of this study could promote a shift from traditional laboratory-based detection to on-site monitoring, facilitating early warning against HABs.


Assuntos
Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/metabolismo , Proliferação Nociva de Algas , Ouro/química , Proteínas Associadas a CRISPR/genética , Endodesoxirribonucleases/genética , Proteínas de Bactérias/genética
8.
Anal Chim Acta ; 1315: 342816, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38879214

RESUMO

BACKGROUND: The monitoring of concentration variation of the newly developed growth differentiation factor 15 (GDF15) biomarker in human serum is of great significance for diagnosing cardiovascular diseases. Current methods for the detection of the GDF15 protein mainly are based on antibody-assisted immunoassays, which encounter the limitations in terms of sensitivity, complexity and costs. The development of simple and sensitive biosensors for GDF15 can therefore facilitate the diagnosis of cardiovascular diseases. RESULTS: A new bimetallic quasi-Cu/Co-MOF nanozyme with high catalytic performance for electrochemical reduction of H2O2 is synthesized via simple one-step precipitation and low-temperature calcination method. Such nanozymes are further employed as amplification tags and coupled with cyclic entropy-driven DNA signal enhancement strategies to construct ultrasensitive aptamer-based biosensor for detecting GDF15 in human serums. GDF15 molecules associate with two aptamers and release the ssDNA trigger sequences via target-binding induced displacement reaction. These ssDNAs subsequently initiate cyclic DNA-fueled strand displacement and catalytic hairpin assembly (CHA) reaction cascades for confining many quasi-Cu/Co-MOF nanozymes on sensor electrode, which yield drastically amplified H2O2 reduction current for detecting GDF15 down to 0.12 pg mL-1 with a dynamic range of 0.5 pg mL-1 to 20 ng mL-1. The electrochemical aptasensor also presents good reproducibility and selectivity and exhibits the capability to detect GDF15 in diluent serums. SIGNIFICANCE: Our aptamer-based GDF15 protein electrochemical assay clearly outperforms current existing antibody-based methods and the quasi-Cu/Co-MOF nanozyme/entropy-driven cascaded signal amplification means can be used as a universal strategy for sensitive monitoring of different biomolecular markers for diverse applications.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Cobalto , Cobre , Técnicas Eletroquímicas , Fator 15 de Diferenciação de Crescimento , Estruturas Metalorgânicas , Aptâmeros de Nucleotídeos/química , Fator 15 de Diferenciação de Crescimento/sangue , Fator 15 de Diferenciação de Crescimento/química , Cobre/química , Humanos , Estruturas Metalorgânicas/química , Cobalto/química , Técnicas Biossensoriais/métodos , Entropia , Peróxido de Hidrogênio/química , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , DNA/química
9.
Trop Biomed ; 41(1): 64-69, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38852135

RESUMO

COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global health threat. Timely identification of infected cases is important for appropriate patient management and the control of viral spread. Simple and cost-effective tests are required to increase access to testing and early case detection. Here, we describe a colorimetric reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method to detect SARS-CoV-2. The RT-LAMP could amplify the orf1ab sequence detectable by visual color change within 45 min at 63 °C. The limit of detection (LoD) for SARS-CoV-2 RNA was less than 100 copies (13.36) per reaction with no cross-amplification with other related viruses. Clinical evaluation using leftover RNA samples extracted from 163 nasopharyngeal swab specimens showed perfect agreement in negative (n = 124) and positive samples with cycle thresholds (Ct) < 34 cycles (n = 33) detected by real-time reverse transcription-polymerase chain reaction (RT-PCR), targeting RdRp and N genes as a reference. Overall, the diagnostic accuracy, sensitivity, specificity, positive and negative predictive values of RT-LAMP in testing were 96.32% (95% CI: 92.16-98.64%), 84.62% (95% CI: 68.47-94.14%), 100% (95% CI: 97.07-100.0%), 100% (95% CI: 89.42-100.0%), and 95.38% (95% CI: 90.22-98.29), respectively. This RT-LAMP assay is simple and reliable, with the potential to be an alternative for the rapid detection of SAR-CoV-2 with minimal time and fewer resources compared to real-time RT-PCR.


Assuntos
COVID-19 , Colorimetria , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , SARS-CoV-2 , Sensibilidade e Especificidade , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Humanos , Tailândia , Colorimetria/métodos , COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , RNA Viral/genética , Transcrição Reversa , Teste de Ácido Nucleico para COVID-19/métodos , Limite de Detecção , Nasofaringe/virologia
10.
Anal Chem ; 96(23): 9424-9429, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38825761

RESUMO

Candida auris (C. auris) was first discovered in Japan in 2009 and has since spread worldwide. It exhibits strong transmission ability, high multidrug resistance, blood infectivity, and mortality rates. Traditional diagnostic techniques for C. auris have shortcomings, leading to difficulty in its timely diagnosis and identification. Therefore, timely and accurate diagnostic assays for clinical samples are crucial. We developed a novel, rapid recombinase-aided amplification (RAA) assay targeting the 18S rRNA, ITS1, 5.8S rRNA, ITS2, and 28S rRNA genes for C. auris identification. This assay can rapidly amplify DNA at 39 °C in 20 min. The analytical sensitivity and specificity were evaluated. From 241 clinical samples collected from pediatric inpatients, none were detected as C. auris-positive. We then prepared simulated clinical samples by adding 10-fold serial dilutions of C. auris into the samples to test the RAA assay's efficacy and compared it with that of real-time PCR. The assay demonstrated an analytical sensitivity of 10 copies/µL and an analytical specificity of 100%. The lower detection limit of the RAA assay for simulated clinical samples was 101 CFU/mL, which was better than that of real-time PCR (102-103 CFU/mL), demonstrating that the RAA assay may have a better detection efficacy for clinical samples. In summary, the RAA assay has high sensitivity, specificity, and detection efficacy. This assay is a potential new method for detecting C. auris, with simple reaction condition requirements, thus helping to manage C. auris epidemics.


Assuntos
Candida auris , Técnicas de Amplificação de Ácido Nucleico , Recombinases , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Recombinases/metabolismo , Candida auris/genética , Candidíase/diagnóstico , Candidíase/microbiologia , Limite de Detecção , DNA Fúngico/genética , DNA Fúngico/análise
11.
J Med Virol ; 96(6): e29744, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38874258

RESUMO

Ebolavirus disease (EVD) is an often-lethal disease caused by the genus Ebolavirus (EBOV). Although vaccines are being developed and recently used, outbreak control still relies on a combination of various factors, including rapid identification of EVD cases. This allows rapid patient isolation and control measure implementation. Ebolavirus diagnosis is performed in treatment centers or reference laboratories, which usually takes a few hours to days to confirm the outbreak or deliver a clear result. A fast and field-deployable molecular detection method, such as the isothermal amplification recombinase-aided amplification (RAA), could significantly reduce sample-to-result time. In this study, a RT-RAA assay was evaluated for EBOV detection. Various primer and probe combinations were screened; analytical sensitivity and cross-specificity were tested. A total of 40 archived samples from the 2014 to 2016 Ebola outbreak in West Africa were tested with both the reference method real-time RT-PCR and the established RT-RAA assay. The assay could detect down to 22.6 molecular copies per microliter. No other pathogens were detected with the Ebolavirus RT-RAA assay. Testing 40 samples yield clinical sensitivity and specificity of 100% each. This rapid isothermal RT-RAA assay can replace the previous RT-RPA and continue to offer rapid EBOV diagnostics.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Recombinases , Sensibilidade e Especificidade , Ebolavirus/genética , Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Recombinases/metabolismo , Técnicas de Diagnóstico Molecular/métodos , África Ocidental/epidemiologia , Surtos de Doenças , RNA Viral/genética , Primers do DNA/genética
12.
Biosens Bioelectron ; 260: 116429, 2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-38838573

RESUMO

Developing highly sensitive and specific on-site tests is imperative to strengthen preparedness against future emerging infectious diseases. Here, we describe the construction of a Cas12a-mediated DNAzyme actuator capable of converting the recognition of a specific DNA sequence into an amplified colorimetric signal. To address viral RNA extraction challenges for on-site applications, we developed a rapid and efficient method capable of lysing the viral particles, preserving the released viral RNA, and concentrating the viral RNA. Integration of the DNAzyme actuator with the viral RNA extraction method and loop-mediated isothermal amplification enables a streamlined colorimetric assay for highly sensitive colorimetric detection of respiratory RNA viruses in gargle and saliva. This assay can detect as few as 83 viral particles/100 µL in gargle and 166 viral particles/100 µL in saliva. The entire assay, from sample processing to visual detection, was completed within 1 h at a single controlled temperature. We validated the assay by detecting SARS-CoV-2 in 207 gargle and saliva samples, achieving a clinical sensitivity of 96.3 % and specificity of 100%. The assay is adaptable for detecting specific nucleic acid sequences in other pathogens and is suitable for resource-limited settings.


Assuntos
Técnicas Biossensoriais , Colorimetria , DNA Catalítico , Técnicas de Amplificação de Ácido Nucleico , RNA Viral , SARS-CoV-2 , Saliva , Colorimetria/métodos , RNA Viral/isolamento & purificação , RNA Viral/genética , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/genética , DNA Catalítico/química , Técnicas Biossensoriais/métodos , Saliva/virologia , Saliva/química , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , COVID-19/virologia , COVID-19/diagnóstico , Proteínas Associadas a CRISPR/isolamento & purificação , Proteínas Associadas a CRISPR/química , Endodesoxirribonucleases/química , Limite de Detecção , Fezes/virologia , Fezes/química , Proteínas de Bactérias , Técnicas de Diagnóstico Molecular
13.
BMJ Open ; 14(6): e084806, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862220

RESUMO

INTRODUCTION: Sexually transmitted diseases (STDs) are a major cause of long-term disability. Urethral discharge syndrome (UDS), abnormal vaginal discharge (AVD) and genital ulcer disease (GUD) are very common in low-income and middle-income countries (LMICs), where, due to lack of resources, these infections are managed according to a syndromic approach. Although microbiological diagnosis using nuclear acid amplification tests (NAAT) is already a standard to prescribe targeted treatments in industrialised countries, no randomised clinical trials have been conducted to evaluate clinical usefulness and acceptability of NAAT in comparison with syndromic approach in LMICs. The results of this study could inform diagnostic guidelines since they may suggest an update of the current recommendation if microbiological diagnosis using NAAT in the management of STD is demonstrated to be both useful and acceptable in an LMIC context. METHODS AND ANALYSIS: The primary objective of this randomised, open-label trial is to evaluate the clinical usefulness of a NAAT and its acceptability in comparison with a clinical syndromic approach and to explore whether this test could replace the syndromic approach in the management of STDs at a national referral hospital in Uganda. 220 patients presenting to the STD clinic at Mulago Hospital in Kampala, Uganda with AVD, UDS or GUD will be randomised to either standard of care (syndromic management) or NAAT-based treatment with a 1:1 ratio. All the patients will be asked to return after 2 or 3 weeks for a control visit. Primary outcome will be therapeutic appropriateness. ETHICS AND DISSEMINATION: This trial was approved by the Mulago Hospital Research and Ethical Committee (MHREC2023-97) and the Uganda National Council for Science and Technology (HS31000ES). Patients will give informed consent to participate before taking part in the study. Results will be published in peer-reviewed journals in open-access formats and data made available in anonymised form. TRIAL REGISTRATION NUMBER: NCT05994495.


Assuntos
Técnicas de Amplificação de Ácido Nucleico , Infecções Sexualmente Transmissíveis , Humanos , Uganda , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções Sexualmente Transmissíveis/diagnóstico , Infecções Sexualmente Transmissíveis/terapia , Feminino , Masculino , Adulto , Ensaios Clínicos Controlados Aleatórios como Assunto , Descarga Vaginal/microbiologia , Descarga Vaginal/diagnóstico , Adolescente
14.
Sex Transm Dis ; 51(6): 388-392, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38733972

RESUMO

BACKGROUND: Standard-of-care nucleic acid amplification tests (routine NAATs) for Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT) can take several days to result and therefore delay treatment. Rapid point-of-care GC/CT NAAT (rapid NAAT) could reduce the time to treatment and therefore onward transmission. This study evaluated the incremental cost per infectious day averted and overall cost of implementation associated with rapid compared with routine NAAT. METHODS: Prospective sexually transmitted infection (STI) treatment data from men who have sex with men and transgender women in San Diego who received rapid NAAT between November 2018 and February 2021 were evaluated. Historical time from testing to treatment for routine NAAT was abstracted from the literature. Costs per test for rapid and routine NAAT were calculated using a micro-costing approach. The incremental cost per infectious day averted comparing rapid to routine NAAT and the costs of rapid GC/CT NAAT implementation in San Diego Public Health STI clinics were calculated. RESULTS: Overall, 2333 individuals underwent rapid NAAT with a median time from sample collection to treatment of 2 days compared with 7 to 14 days for routine NAAT equating to a reduction of 5 to 12 days. The cost of rapid and routine GC/CT NAAT was $57.86 and $18.38 per test, respectively, with a cost-effectiveness of between $2.43 and $5.82 per infectious day averted. The incremental cost of rapid NAAT improved when at least 2000 tests were performed annually. CONCLUSIONS: Although rapid GC/CT NAAT is more expensive than routine testing, the reduction of infectious days between testing and treatment may reduce transmission and provide improved STI treatment services to patients.


Assuntos
Infecções por Chlamydia , Chlamydia trachomatis , Gonorreia , Homossexualidade Masculina , Neisseria gonorrhoeae , Técnicas de Amplificação de Ácido Nucleico , Humanos , Masculino , Gonorreia/diagnóstico , Gonorreia/economia , Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Neisseria gonorrhoeae/isolamento & purificação , Chlamydia trachomatis/isolamento & purificação , Adulto , California/epidemiologia , Análise Custo-Benefício , Estudos Prospectivos , Feminino , Testes Imediatos/economia , Pessoas Transgênero
15.
Gene ; 922: 148544, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-38734187

RESUMO

This study introduces an efficient RPA-PfAgo detection system for the MTHFR C677T polymorphism, proposing a potential strategy to simplify the genotyping process. By optimizing recombinase polymerase amplification (RPA) with Pyrococcus furiosus Argonaute (PfAgo) nucleases, we achieved DNA amplification at a constant temperature. The assay was fine-tuned through meticulous primer and guide DNA selection, with optimal conditions established at 2.0 µL of MgAc, a reaction temperature of 42 °C, and a 10-minute reaction time for RPA. Further optimization of the PfAgo cleavage assay revealed the ideal concentrations of MnCl2, guide DNA, molecular beacon probes, the PfAgo enzyme, and the RPA product to maximize sensitivity and specificity. Clinical validation of 20 samples showed 100% concordance with Sanger sequencing, confirming the method's precision. The RPA-PfAgo system is a promising tool for on-site genotyping, with broad applications in personalized medicine and disease prevention.


Assuntos
Técnicas de Genotipagem , Metilenotetra-Hidrofolato Redutase (NADPH2) , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Técnicas de Genotipagem/métodos , Polimorfismo de Nucleotídeo Único , Pyrococcus furiosus/genética , Pyrococcus furiosus/enzimologia , Genótipo , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas Argonautas/genética , Recombinases/metabolismo , Recombinases/genética
16.
Anal Chim Acta ; 1311: 342720, 2024 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-38816155

RESUMO

BACKGROUND: The monkeypox virus (MPXV) is a linear double-stranded DNA virus with a large genome that causes tens of thousands of infections and hundreds of deaths in at least 40 countries and regions worldwide. Therefore, timely and accurate diagnostic testing could be an important measure to prevent the ongoing spread of MPXV and widespread epidemics. RESULTS: Here, we designed multiple sets of primers for the target region of MPXV for loop-mediated isothermal amplification (LAMP) detection and identified the optimal primer set. Then, the specificity in fluorescent LAMP detection was verified using the plasmids containing the target gene, pseudovirus and other DNA/RNA viruses. We also evaluated the sensitivity of the colorimetric LAMP detection system using the plasmid and pseudovirus samples, respectively. Besides, we used monkeypox pseudovirus to simulate real samples for detection. Subsequent to the establishment and introduction of a magnetic beads (MBs)-based nucleic acid extraction technique, an integrated device was developed, characterized by rapidity, high sensitivity, and remarkable specificity. This portable system demonstrated a visual detection limit of 137 copies/mL, achieving sample-to-answer detection within 1 h. SIGNIFICANCE: The device has the advantages of integration, simplicity, miniaturization, and visualization, which help promote the realization of accurate, rapid, portable, and low-cost testing. Meanwhile, this platform could facilitate efficient, cost-effective and easy-operable point-of-care testing (POCT) in diverse resource-limited settings in addition to the laboratory.


Assuntos
Colorimetria , Monkeypox virus , Técnicas de Amplificação de Ácido Nucleico , Colorimetria/métodos , Colorimetria/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Monkeypox virus/genética , Monkeypox virus/isolamento & purificação , Limite de Detecção , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/instrumentação
17.
Adv Exp Med Biol ; 1451: 253-271, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38801583

RESUMO

An outbreak of monkeypox (Mpox) was reported in more than 40 countries in early 2022. Accurate diagnosis of Mpox can be challenging, but history, clinical findings, and laboratory diagnosis can establish the diagnosis. The pre-analytic phase of testing includes collecting, storing, and transporting specimens. It is advised to swab the lesion site with virus transport medium (VTM) containing Dacron or polyester flock swabs from two different sites. Blood, urine, and semen samples may also be used. Timely sampling is necessary to obtain a sufficient amount of virus or antibodies. The analytical phase of infectious disease control involves diagnostic tools to determine the presence of the virus. While polymerase chain reaction (PCR) is the gold standard for detecting Mpox, genome sequencing is for identifying new or modified viruses. As a complement to these methods, isothermal amplification methods have been designed. ELISA assays are also available for the determination of antibodies. Electron microscopy is another effective diagnostic method for tissue identification of the virus. Wastewater fingerprinting provides some of the most effective diagnostic methods for virus identification at the community level. The advantages and disadvantages of these methods are further discussed. Post-analytic phase requires proper interpretation of test results and the preparation of accurate patient reports that include relevant medical history, clinical guidelines, and recommendations for follow-up testing or treatment.


Assuntos
Mpox , Humanos , Mpox/diagnóstico , Mpox/virologia , Mpox/epidemiologia , Monkeypox virus/genética , Monkeypox virus/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Manejo de Espécimes/métodos , Técnicas de Laboratório Clínico/métodos
18.
Cells ; 13(10)2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38786028

RESUMO

Zika (ZIKV) and Chikungunya (CHIKV) viruses are mosquito-transmitted infections, or vector-borne pathogens, that emerged a few years ago. Reliable diagnostic tools for ZIKV and CHIKV-inexpensive, multiplexed, rapid, highly sensitive, and specific point-of-care (POC) systems-are vital for appropriate risk management and therapy. We recently studied a detection system with great success in Mexico (Villahermosa, state of Tabasco), working with human sera from patients infected with those viruses. The research conducted in Mexico validated the efficacy of a novel two-step rapid isothermal amplification technique (RAMP). This approach, which encompasses recombinase polymerase amplification (RPA) followed by loop-mediated isothermal amplification (LAMP), had been previously established in the lab using lab-derived Zika (ZIKV) and Chikungunya (CHIKV) viruses. Crucially, our findings confirmed that this technique is also effective when applied to human sera samples collected from locally infected individuals in Mexico.


Assuntos
Vírus Chikungunya , Técnicas de Amplificação de Ácido Nucleico , Infecção por Zika virus , Zika virus , Humanos , Zika virus/genética , Zika virus/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Vírus Chikungunya/genética , Vírus Chikungunya/isolamento & purificação , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologia , Infecção por Zika virus/sangue , Febre de Chikungunya/diagnóstico , Febre de Chikungunya/virologia , Febre de Chikungunya/sangue , Técnicas de Diagnóstico Molecular/métodos , RNA Viral/genética , RNA Viral/sangue , México , Sensibilidade e Especificidade , Vírus de RNA/genética , Vírus de RNA/isolamento & purificação
19.
BMC Vet Res ; 20(1): 203, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38755641

RESUMO

BACKGROUND: Avian influenza virus (AIV) not only causes huge economic losses to the poultry industry, but also threatens human health. Reverse transcription recombinase-aided amplification (RT-RAA) is a novel isothermal nucleic acid amplification technology. This study aimed to improve the detection efficiency of H5, H7, and H9 subtypes of AIV and detect the disease in time. This study established RT-RAA-LFD and real-time fluorescence RT-RAA (RF-RT-RAA) detection methods, which combined RT-RAA with lateral flow dipstick (LFD) and exo probe respectively, while primers and probes were designed based on the reaction principle of RT-RAA. RESULTS: The results showed that RT-RAA-LFD could specifically amplify H5, H7, and H9 subtypes of AIV at 37 °C, 18 min, 39 °C, 20 min, and 38 °C, 18 min, respectively. The sensitivity of all three subtypes for RT-RAA-LFD was 102 copies/µL, which was 10 ∼100 times higher than that of reverse transcription polymerase chain reaction (RT-PCR) agarose electrophoresis method. RF-RT-RAA could specifically amplify H5, H7, and H9 subtypes of AIV at 40 °C, 20 min, 38 °C, 16 min, and 39 °C, 17 min, respectively. The sensitivity of all three subtypes for RF-RT-RAA was 101 copies/µL, which was consistent with the results of real-time fluorescence quantification RT-PCR, and 100 ∼1000 times higher than that of RT-PCR-agarose electrophoresis method. The total coincidence rate of the two methods and RT-PCR-agarose electrophoresis in the detection of clinical samples was higher than 95%. CONCLUSIONS: RT-RAA-LFD and RF-RT-RAA were successfully established in this experiment, with quick response, simple operation, strong specificity, high sensitivity, good repeatability, and stability. They are suitable for the early and rapid diagnosis of Avian influenza and they have positive significance for the prevention, control of the disease, and public health safety.


Assuntos
Galinhas , Vírus da Influenza A , Influenza Aviária , Técnicas de Amplificação de Ácido Nucleico , Recombinases , Transcrição Reversa , Animais , Influenza Aviária/virologia , Influenza Aviária/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/veterinária , Técnicas de Amplificação de Ácido Nucleico/métodos , Vírus da Influenza A/genética , Vírus da Influenza A/classificação , Vírus da Influenza A/isolamento & purificação , Recombinases/metabolismo , Sensibilidade e Especificidade , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/diagnóstico
20.
J Agric Food Chem ; 72(22): 12788-12797, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38778779

RESUMO

Fish from the pike (Esox) genus are valued in gastronomy for their superior meat quality. However, they can cause allergic reactions in sensitive consumers. This work aimed to fill the gap in the detection of pike allergens using molecular-biological techniques. New, fast, and accurate loop-mediated isothermal amplification (LAMP) and real-time PCR (qPCR) assays were designed to detect pike DNA using the parvalbumin gene as a marker. LAMP was assessed by electrophoresis, SYBR green optical detection, and real-time fluorescence detection. The latter was the most sensitive, detecting as little as 0.78 ng of pike DNA; the qPCR detection limit was 0.1 ng. The LAMP analysis took 20-70 min, which is significantly faster than qPCR. The study provides reliable detection and quantification of the parvalbumin gene in both fresh and processed samples and further highlights the versatility of the use of the parvalbumin gene for the authentication of food products and consumer protection via refined allergen risk assessment that is independent of the type of tissue or food processing method used.


Assuntos
Alérgenos , Esocidae , Hipersensibilidade Alimentar , Parvalbuminas , Parvalbuminas/genética , Parvalbuminas/imunologia , Parvalbuminas/análise , Alérgenos/genética , Alérgenos/análise , Alérgenos/imunologia , Animais , Hipersensibilidade Alimentar/imunologia , Esocidae/genética , Esocidae/imunologia , Medição de Risco , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Humanos , Contaminação de Alimentos/análise , Biomarcadores/análise , Técnicas de Diagnóstico Molecular
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