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1.
Biomed Res Int ; 2021: 6653950, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34124257

RESUMO

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 µL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 µL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng/uL, which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.


Assuntos
COVID-19/diagnóstico , Proteínas do Envelope de Coronavírus/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , 2-Propanol/química , Biomarcadores/análise , COVID-19/virologia , Primers do DNA/síntese química , Primers do DNA/genética , Humanos , Nasofaringe/virologia , Orofaringe/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
2.
Biomed Res Int ; 2021: 6653950, 2021.
Artigo em Inglês | MEDLINE | ID: covidwho-1263958

RESUMO

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 µL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 µL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng/uL, which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.


Assuntos
COVID-19/diagnóstico , Proteínas do Envelope de Coronavírus/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , 2-Propanol/química , Biomarcadores/análise , COVID-19/virologia , Primers do DNA/síntese química , Primers do DNA/genética , Humanos , Nasofaringe/virologia , Orofaringe/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
3.
J Virol Methods ; 294: 114171, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33984394

RESUMO

Respiratory syncytial virus (RSV) is a common cause of acute respiratory disease worldwide, especially in young children. The World Health Organization (WHO) has initiated an RSV Surveillance Pilot program that aims to perform worldwide RSV surveillance, requiring the development of reliable and rapid molecular methods to detect and identify RSV. A duplex real-time RT-PCR assay developed for simultaneous detection of both A and B subtypes of RSV was included as part of this program. This duplex assay targeted a conserved region of the RSV polymerase gene and was validated for analytical sensitivity, specificity, reproducibility and clinical performance with a wide range of respiratory specimens. The assay was highly specific for RSV and did not react with non-RSV respiratory pathogens, including the SARS-CoV-2 virus.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , RNA Viral/isolamento & purificação , Vírus Sincicial Respiratório Humano/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Primers do DNA/genética , Humanos , Limite de Detecção , Nasofaringe/virologia , RNA Polimerase Dependente de RNA/genética , Reprodutibilidade dos Testes , Ribonuclease P/genética , SARS-CoV-2/genética , Sensibilidade e Especificidade
4.
J Virol Methods ; 294: 114171, 2021 08.
Artigo em Inglês | MEDLINE | ID: covidwho-1226315

RESUMO

Respiratory syncytial virus (RSV) is a common cause of acute respiratory disease worldwide, especially in young children. The World Health Organization (WHO) has initiated an RSV Surveillance Pilot program that aims to perform worldwide RSV surveillance, requiring the development of reliable and rapid molecular methods to detect and identify RSV. A duplex real-time RT-PCR assay developed for simultaneous detection of both A and B subtypes of RSV was included as part of this program. This duplex assay targeted a conserved region of the RSV polymerase gene and was validated for analytical sensitivity, specificity, reproducibility and clinical performance with a wide range of respiratory specimens. The assay was highly specific for RSV and did not react with non-RSV respiratory pathogens, including the SARS-CoV-2 virus.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , RNA Viral/isolamento & purificação , Vírus Sincicial Respiratório Humano/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Primers do DNA/genética , Humanos , Limite de Detecção , Nasofaringe/virologia , RNA Polimerase Dependente de RNA/genética , Reprodutibilidade dos Testes , Ribonuclease P/genética , SARS-CoV-2/genética , Sensibilidade e Especificidade
5.
Acta Trop ; 220: 105958, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34004173

RESUMO

Plague is a zoonotic disease caused by Yersinia pestis, a Gram-negative, rod shaped coccobacillus, which is primarily found in rodents and can be transmitted to humans through flea bite. The disease has three major clinical forms bubonic (by flea bite), pneumonic (by respiratory droplets) and septicemic plague. Y. pestis is classified as a category 'A' agent by NIAID, USA due to its high mortality and easy person to person dissemination. The conventional diagnostic methods available for Y. pestis show cross-reactivity with other enteropathogenic bacteria making its detection difficult. There is a need to develop sensitive and specific molecular assay for accurate detection of Y. pestis. PCR is well suited molecular biology tool for rapid diagnosis of plague but after completion of thermal cycling steps, it requires additional time to analyze amplified product using agarose gel electrophoresis. In the present study, PCR assay coupled with lateral flow strips has been developed for rapid detection of Y. pestis. Lateral flow strips give an alternative to gel electrophoresis and permit easy and rapid detection of PCR products. The PCR was performed with 5' 6-FAM and biotin tagged primers specific for Y. pestis, targeting yihN gene located on chromosome. The PCR product was analyzed using lateral flow strips which yielded result within 2-3 minutes. The analytical sensitivity of PCR-lateral flow (PCR-LF) assay was 1 pg genomic DNA of Y. pestis and 500 copies of target DNA sequence harboured in a recombinant plasmid. The assay could detect Y. pestis DNA extracted from spiked human blood samples containing ≥104 CFU per mL of bacteria. The assay was found to be specific and did not cross react with other closely related bacterial species. The developed assay was highly specific, sensitive and also did not require agarose gel electrophoresis for post amplification analysis.


Assuntos
Peste/microbiologia , Reação em Cadeia da Polimerase/métodos , Yersinia pestis/genética , Yersinia pestis/isolamento & purificação , Animais , Sequência de Bases , Primers do DNA/genética , Humanos , Yersinia pestis/fisiologia
6.
Microbes Environ ; 36(2)2021.
Artigo em Inglês | MEDLINE | ID: mdl-33952860

RESUMO

Fragmented and primer ligated dsRNA sequencing (FLDS) is a sequencing method applicable to long double-stranded RNA (dsRNA) that enables the complete genome sequencing of both double- and single-stranded RNA viruses. However, the application of this method on a low amount of dsRNA has been hindered by adaptor dimer formation during cDNA amplification and sequence library preparation. We herein developed FLDS ver. 3 by optimizing the terminal modification of an oligonucleotide adaptor and the conditions of adaptor ligation. We also examined the concentration of Mg2+ in the PCR reaction for cDNA amplification and the purification method of amplified cDNA. Fine sequence reads were successfully obtained from metagenomic shotgun sequencing libraries constructed from 10 and 100 pg dsRNA, and these libraries exhibited weaker detection sensitivity for low-abundance dsRNAs (viral genomes and genome segments) than that constructed from 1| |ng of dsRNA. We also report the utility of capillary electrophoresis for dsRNA quantification. The FLDS ver. 3 package expands the frontiers of our knowledge in RNA virus diversity and evolution.


Assuntos
Vírus de RNA/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética , Análise de Sequência de RNA/métodos , Primers do DNA/genética , Genoma Viral , Metagenômica , Reação em Cadeia da Polimerase , Vírus de RNA/isolamento & purificação
7.
Methods Mol Biol ; 2281: 323-332, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847969

RESUMO

The single-stranded DNA-binding protein gp2.5 of bacteriophage T7 plays myriad functions in the replication of phage genomes. In addition to interacting with ssDNA, gp2.5 binds to the T7 DNA polymerase and primase/helicase proteins, regulating their enzymatic activities. Here we describe in vitro methods to examine the effects of gp2.5 on primer synthesis and extension by the T7 replisome.


Assuntos
Bacteriófago T7/fisiologia , Primers do DNA/síntese química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/metabolismo , Primers do DNA/genética , Replicação do DNA , DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Replicação Viral
8.
Int J Infect Dis ; 107: 145-152, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33864913

RESUMO

OBJECTIVE: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and it remains a significant health problem in several parts of the world. Early and accurate diagnosis of this disease is therefore essential. Previously published loop-mediated isothermal amplification (LAMP) protocols for detecting mycobacterial species used conventional primers targeting the 16S rRNA, gyrB and insertion sequence genes. METHODS: In this study, we conducted a LAMP assay for leprosy and compared it with quantitative polymerase chain reaction (q-PCR) and conventional PCR assays to determine the efficiency, sensitivity and specificity of each technique. We chose conserved sequence RLEP as a suitable molecular target for assays. RESULTS: The LAMP assay provided rapid and accurate results, confirming leprosy in 91/110 clinical skin tissue samples from leprosy patients and amplifying the target pathogen in <60 min at 65 °C. The assay was more sensitive than conventional PCR and more straightforward and faster than the q-PCR assay. CONCLUSIONS: The LAMP assay has the potential for developing quicker, more accessible visual methods for the detection of M. leprae, which will enable early diagnosis and treatment and prevent further infection in endemic areas.


Assuntos
Hanseníase/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium leprae/genética , Mycobacterium leprae/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Primers do DNA/genética , Humanos , Limite de Detecção , Mycobacterium leprae/fisiologia , RNA Ribossômico 16S/genética
9.
Sci Rep ; 11(1): 8988, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: covidwho-1203449

RESUMO

Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.


Assuntos
Primers do DNA/genética , Sondas de DNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Genoma Viral , Mutação
10.
Methods Mol Biol ; 2250: 245-256, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33900610

RESUMO

Retrotransposons are ubiquitous, generally dispersed components of eukaryotic genomes. These properties, together with their "copy and paste" lifecycle that generates insertional polymorphism without need for excision, makes them widely useful as a molecular-genetic tags. Various tagging systems have been developed that exploit the sequence conservation of retrotransposon components, such as those found in their long terminal repeats (LTRs). To detect polymorphisms for retrotransposon insertions, marker systems generally rely on PCR amplification between the termini and some component of flanking genomic DNA. As complements to various "wet lab" protocols for retrotransposon tagging, in silico bioinformatics approaches are useful for predicting likely outcomes from unsequenced accessions on the basis of reference genomes. In this chapter, we describe protocols for in silico retrotransposon-based fingerprinting techniques using the FastPCR software as an integrated tools environment for in silico PCR primer design and analysis.


Assuntos
Biologia Computacional/métodos , Simulação por Computador , Reação em Cadeia da Polimerase/métodos , Retroelementos/genética , Software , Impressões Digitais de DNA/métodos , Primers do DNA/genética , Internet , Repetições de Microssatélites/genética , Polimorfismo Genético
11.
Food Chem ; 355: 129525, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-33799266

RESUMO

Available nuclear gene sequences for meat detection are still rare and little applicability in the investigation of new types of meat adulteration such as fox, mink and raccoon dog was performed. In the present work, we developed a reliable qualitative and quantitative detection method for fur-bearing animal meat based on droplet digital PCR (ddPCR). Three sets of primers and probes targeted nuclear genes for fox, mink and raccoon dog were designed for ddPCR system; In addition, turkey was selected as internal reference to transform the copy numbers to the fraction of target species. Results indicated that the dynamic ranges of three fur-bearing animals were all from 1% to 90%; the limit of detection (LOD) and limit of quantification (LOQ) for three fur-bearing animals were same, with LOD 0.1% (w/w) and LOQ 1% (w/w). Moreover, we confirmed that different additives had no effect on quantification accuracy in the ddPCR assay.


Assuntos
Pelo Animal , Análise de Alimentos/métodos , Manipulação de Alimentos , Carne/análise , Reação em Cadeia da Polimerase/métodos , Animais , Primers do DNA/genética , Limite de Detecção , Mamíferos
12.
Sci Rep ; 11(1): 8988, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33903676

RESUMO

Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.


Assuntos
Primers do DNA/genética , Sondas de DNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Genoma Viral , Mutação
13.
Vopr Virusol ; 66(1): 17-28, 2021 03 07.
Artigo em Russo | MEDLINE | ID: covidwho-1121949

RESUMO

This review presents the basic principles of application of the loop-mediated isothermal amplification (LAMP) reaction for the rapid diagnosis of coronavirus infection caused by SARS-CoV-2. The basic technical details of the method, and the most popular approaches of specific and non-specific detection of amplification products are briefly described. We also discuss the first published works on the use of the method for the detection of the nucleic acid of the SARS-CoV-2 virus, including those being developed in the Russian Federation. For commercially available and published LAMP-based assays, the main analytical characteristics of the tests are listed, which are often comparable to those based on the method of reverse transcription polymerase chain reaction (RT-PCR), and in some cases are even superior. The advantages and limitations of this promising methodology in comparison to other methods of molecular diagnostics, primarily RT-PCR, are discussed, as well as the prospects for the development of technology for the detection of other infectious agents.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificação de Ácido Nucleico/normas , RNA Viral/genética , SARS-CoV-2/genética , Artefatos , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/normas , Primers do DNA/genética , Primers do DNA/metabolismo , Sondas de DNA/genética , Sondas de DNA/metabolismo , Humanos , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade
14.
PLoS One ; 16(3): e0248042, 2021.
Artigo em Inglês | MEDLINE | ID: covidwho-1115310

RESUMO

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).


Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , COVID-19/genética , Teste para COVID-19/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Primers do DNA/genética , Humanos , Proteínas do Nucleocapsídeo/genética , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa/genética , SARS-CoV-2/patogenicidade , Sensibilidade e Especificidade
15.
PLoS One ; 16(3): e0248042, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33657176

RESUMO

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).


Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , COVID-19/genética , Teste para COVID-19/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Primers do DNA/genética , Humanos , Proteínas do Nucleocapsídeo/genética , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa/genética , SARS-CoV-2/patogenicidade , Sensibilidade e Especificidade
16.
Int J Food Microbiol ; 344: 109113, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33652337

RESUMO

Ochratoxins are a group of mycotoxins that frequently occur as contaminants in agricultural commodities and foods, including dry-cured meats and cheeses. The fungus Aspergillus westerdijkiae is frequently isolated from aged foods and can produce ochratoxin A (OTA). However, individual strains of the fungus can have one of two OTA production phenotypes (chemotypes): OTA production and OTA nonproduction. Monitoring and early detection of OTA-producing fungi in food are the most effective strategies to manage OTA contamination. Therefore, we examined genome sequence data from five A. westerdijkiae strains isolated from the surface of cheese from southern Italy to identify genetic markers indicative of the twoOTA chemotypes. This analysis revealed a naturally occurring deletion of the OTA regulatory gene, otaR, in an OTA-nonproducing isolate.We used this information to design a polymerase chain reaction (PCR) method that could identify A. westerdijkiae and distinguish between the two OTA chemotypes. In this method, the PCR primers were complementary to conserved sequences flanking otaR and yielded different-sized amplicons from strains with the different chemotypes. The primers did not yield ota-region-specific amplicons from other OTA-producing species. Because the method is specific to A. westerdijkiae and can distinguish between the two OTA chemotypes, it has potential to significantly improve OTA monitoring programs.


Assuntos
Aspergillus/metabolismo , Queijo/microbiologia , Alimentos em Conserva/microbiologia , Carne/microbiologia , Ocratoxinas/biossíntese , Reação em Cadeia da Polimerase/métodos , Aspergillus/genética , Aspergillus/isolamento & purificação , Primers do DNA/genética , Contaminação de Alimentos/análise , Itália
17.
Food Chem ; 351: 129348, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-33647699

RESUMO

Adulteration of food ingredients, particularly replacement of high-value milk with low-cost milk, affects food safety. For rapid and accurate identification of the possible adulterating milk species in an unknown sample, a centrifugal microfluidic chip-based real-time fluorescent multiplex loop-mediated isothermal amplification (LAMP) assay was developed to simultaneously detect milk from cow, camel, horse, goat, and yak. Using precoated primers in different reaction wells, the centrifugal microfluidic chip markedly simplified the detection process and reduced false-positive results. The entire amplification was completed within 90 min with a genomic detection limit of 0.05 ng/µL in cow, camel, horse, and goat milk and 0.005 ng/µL in yak milk. Using simulated adulterated samples for validation, the detection limit for adulterated milk samples was 2.5%, satisfying authentication requirements, as the proportion of adulterated milk higher than 10% affects economic interests. Therefore, this simple, centrifugal, microfluidic chip-based multiplex real-time fluorescent LAMP assay can simultaneously detect common milk species in commercial products to enable accurate labeling.


Assuntos
Centrifugação/instrumentação , Qualidade dos Alimentos , Dispositivos Lab-On-A-Chip , Leite/química , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Animais , Bovinos , Primers do DNA/genética , Feminino , Leite/normas , Fatores de Tempo
18.
Vopr Virusol ; 66(1): 17-28, 2021 03 07.
Artigo em Russo | MEDLINE | ID: mdl-33683062

RESUMO

This review presents the basic principles of application of the loop-mediated isothermal amplification (LAMP) reaction for the rapid diagnosis of coronavirus infection caused by SARS-CoV-2. The basic technical details of the method, and the most popular approaches of specific and non-specific detection of amplification products are briefly described. We also discuss the first published works on the use of the method for the detection of the nucleic acid of the SARS-CoV-2 virus, including those being developed in the Russian Federation. For commercially available and published LAMP-based assays, the main analytical characteristics of the tests are listed, which are often comparable to those based on the method of reverse transcription polymerase chain reaction (RT-PCR), and in some cases are even superior. The advantages and limitations of this promising methodology in comparison to other methods of molecular diagnostics, primarily RT-PCR, are discussed, as well as the prospects for the development of technology for the detection of other infectious agents.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificação de Ácido Nucleico/normas , RNA Viral/genética , SARS-CoV-2/genética , Artefatos , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/normas , Primers do DNA/genética , Primers do DNA/metabolismo , Sondas de DNA/genética , Sondas de DNA/metabolismo , Humanos , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade
19.
Appl Microbiol Biotechnol ; 105(5): 1991-2002, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33576884

RESUMO

Black rot and bacterial spots threaten the cultivation of cruciferous vegetables worldwide, and the development of a method that can easily detect, identify, and distinguish their respective pathogens Xanthomonas campestris pv. campestris (Xcc) and X. campestris pv. raphani (Xcr) is required. Multiple whole-genome sequences of Xcc and Xcr were aligned to identify specific regions and subsequently design gene markers. A region present in Xcr, but absent in Xcc, was detected, which was approximately 11.5 kbp in length, sandwiched between the serine protease homolog (SPH) and nicotinate phosphoribosyltransferase gene (pncB). It contained putative cellulose synthesis-related genes, whereas Xcc only had a modified cellulose synthase gene. Designed primers were pncB_fw1 and pncB_fw2 (from the pncB gene), Xcc_rv1 and Xcc_rv2 (from the modified cellulose synthesis gene), and Xcr_rv1 and Xcr_rv2 (from the putative first and second open reading frames of the gene cluster). PCR using pncB_fw1 and Xcc_rv1, or pncB_fw2 and Xcc_rv2, amplified DNA fragments only in Xcc and X. campestris pv. incanae (Xci). Xci is the causal agent of black rot of garden stock and closely related to Xcc. PCR using pncB_fw1 and Xcr_rv1, or pncB_2 and Xcr_rv2, amplified DNA fragments only in Xcr. Multiplex PCR analysis easily distinguished Xcc and Xcr from bacterial colonies isolated on growth media and detected the pathogen in symptomatic leaves. Multiplex nested PCR detected the contamination of one seed with Xcc and/or Xcr infection from 1000 seeds. Therefore, the PCR primers designed in this study therefore helped detect and discriminate between Xcc and Xcr. KEY POINTS: • Xanthomonas campestris pv. campestris (Xcc) and pv. raphani (Xcr) were investigated. • Novel primers were designed following whole-genome comparison analyses. • Multiplex PCR with new primers distinguished Xcc and Xcr simultaneously.


Assuntos
Xanthomonas campestris , Primers do DNA/genética , Marcadores Genéticos , Família Multigênica , Reação em Cadeia da Polimerase Multiplex , Doenças das Plantas , Xanthomonas campestris/genética
20.
PLoS One ; 16(2): e0240002, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33626057

RESUMO

A broad panel of potentially amplifiable microsatellite loci and a multiplex system were developed for the Amazonian symbol fish species Arapaima gigas, which is currently in high danger of extinction due to the disorderly fishing exploitation. Several factors have contributed to the increase of this threat, among which we highlight the lack of genetic information about the structure and taxonomic status of the species, as well as the lack of accurate tools for evaluation of the effectivity of current management programs. Based on Arapaima gigas' whole genome, available at the NCBI database (ID: 12404), a total of 95,098 unique perfect microsatellites were identified, including their proposed primers. From this panel, a multiplex system containing 12 tetranucleotide microsatellite markers was validated. These tools are valuable for research in as many areas as bioinformatics, ecology, genetics, evolution and comparative studies, since they are able to provide more accurate information for fishing management, conservation of wild populations and genetic management of aquaculture.


Assuntos
Conservação dos Recursos Naturais/métodos , Peixes/classificação , Peixes/genética , Animais , Primers do DNA/genética , Espécies em Perigo de Extinção/tendências , Variação Genética/genética , Genoma/genética , Genômica/métodos , Repetições de Microssatélites/genética , Rios , América do Sul
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