Can quantitative RT-PCR for SARS-CoV-2 help in better management of patients and control of coronavirus disease 2019 pandemic
The emergence of SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), represents a public health emergency of unprecedented proportion. The global containment efforts have been focused on testing, tracing of contacts and treatment (isolation) of those found COVID-19 positive. Since the whole genome sequences of a number of strains of this novel RNA virus were available in the public domain by early January 2020, a number of real-time polymerase chain reaction (RT-PCR) protocols were designed and used for diagnosis of this infection.
Most RT-PCRs are designed for qualitative COVID-19 reporting (SARS-CoV-2 detected or not detected), but have been used for semi-quantitative estimation of viral load based on cycle threshold value. Our manuscript discusses the utility of quantitative PCR testing for COVID-19 and its patient management benefits.
Multiplex TaqMan Real-Time PCR Assay for Sensitive Detection of Two Weevil Species (Coleoptera: Curculionidae)
Rapid and cost-efficient identification of Naupactus species is becoming a key process for the exportation of citrus fruit from Chile and other countries, considering the quarantine regulations for some species of the cosmopolitan genus Naupactus.
This study deals with the development of a fast and sensitive detection protocol for Naupactus cervinus (Coleoptera: Curculionidae) (Boheman) and Naupactus xanthographus (Coleoptera: Curculionidae) (Germar) based on multiplex TaqMan Real-time polymerase chain reaction. Both N. cervinus and N. xanthographus primer and probe sets achieved species-specific detection in a linear range from 1 pg/μl to 1 × 10-6 pg/μl, allowing detection of as few as 160 copies of template DNA. Non-target amplifications were not detected and a panel composed of 480 test samples had 100% coincidence with the respective morphological identification.
Validation of swab sampling and SYBR Green-based real-time PCR for the diagnosis of Cutaneous Leishmaniasis in French Guiana
Recent studies have highlighted the interest of non-invasive sampling procedures coupled to real-time PCR methods for detection of Leishmania species in South America. In French Guiana, sampling method still relied on skin biopsies. Non-invasive protocols should be tested on a large annual cohort to improve routine laboratory diagnosis of Cutaneous Leishmaniasis.
Therefore, we evaluated the performances of a new Leishmania detection and species identification protocol involving cotton swabs and SYBR Green real-time PCR of Hsp70 gene, coupled with Sanger sequencing. Between May 2017 and May 2018, 145 patients with ulcerated lesions compatible with Cutaneous Leishmaniasis were included in the Cayenne Hospital and its remote health centres. Each patient underwent scrapings for smear, skin biopsies for parasite culture and PCR-RFLP (RNA pol II) and cotton swabs for SYBR Green PCR. The most accurate diagnostic test was the SYBR Green PCR on swab sampling, showing a 98% sensitivity.
Description: 2x SYBR Green qPCR master mix (High ROX) utilizes a special performance-enhanced Taq DNA polymerase protected via a hot-start activation technique, and optimized qPCR buffer system to perform SYBR
Description: 2x SYBR Green qPCR master mix (High ROX) utilizes a special performance-enhanced Taq DNA polymerase protected via a hot-start activation technique, and optimized qPCR buffer system to perform SYBR
Description: 2x SYBR Green qPCR master mix (Low ROX) utilizes a special performance-enhanced Taq DNA polymerase protected via a hot-start activation technique, and optimized qPCR buffer system to perform SYBR
Description: 2x SYBR Green qPCR master mix (Low ROX) utilizes a special performance-enhanced Taq DNA polymerase protected via a hot-start activation technique, and optimized qPCR buffer system to perform SYBR
To order instruments in 115V / US plug please delete the 'E' off the order code.European 2 pin plugs will be supplied as standard, please request UK if required.
To order instruments in 115V / US plug please delete the 'E' off the order code.European 2 pin plugs will be supplied as standard, please request UK if required.
To order instruments in 115V / US plug please delete the 'E' off the order code.European 2 pin plugs will be supplied as standard, please request UK if required.
To order instruments in 115V / US plug please delete the 'E' off the order code.European 2 pin plugs will be supplied as standard, please request UK if required.
To order instruments in 115V / US plug please delete the 'E' off the order code.European 2 pin plugs will be supplied as standard, please request UK if required.
To order instruments in 115V / US plug please delete the 'E' off the order code.European 2 pin plugs will be supplied as standard, please request UK if required.
Mean PCR cycle threshold (Ct) was of 24.4 Ct (min=17 Ct, max=36 Ct) and was inferior to 35 Ct in 97.6% of samples. All samples positive with real-time PCR SYBR Green were successfully identified at the species level by DNA sequencing. This new method should be considered for routine diagnosis of Cutaneous Leishmaniasis in South America and especially for remote areas, as non-invasive collection tools are easier to use and require less precautions for transportation.
Development and clinical validation of a pangenotypic PCR-based assay for the detection and quantification of hepatitis E virus (Orthopevirus A genus)
The objective of this study was to design a pangenotypic PCR-based assay for the detection and quantification of hepatitis E virus (HEV) RNA from across the entire spectrum of described genotypes belonging to the Orthohepevirus A genus. The optimal conditions and the performance of the assay were determined by testing the WHO standard strain (6219/10) and the WHO HEV panel (8578/13).
Similarly, performance comparisons were made with two commercial assays (Real Star HEV RT-PCR 2.0 and ampliCube HEV 2.0 Quant) to detect HEV RNA at concentrations below 1,000 IU/mL with viral strains from the WHO and to test samples from 54 patients with acute hepatitis. The assay presented in this study was able to detect the entire spectrum of described genotypes belonging to the Orthohepevirus A genus, demonstrating a better performance than both commercial kits. This procedure may represent a significant improvement in the molecular diagnosis of HEV infection.
SARS-CoV-2 and RT-PCR in asymptomatic patients: Results of a cohort of workers at El Dorado International Airport in Bogotá, 2020
Introduction: The 2019 coronavirus pandemic (COVID-19) has caused around 25 million cases worldwide. Asymptomatic patients have been described as potential sources of transmission. However, there are difficulties to detect them and to establish their role in the dynamics of virus transmission, which hinders the implementation of prevention strategies. Objective: To describe the behavior of asymptomatic SARS-CoV-2 virus infection in a cohort of workers at the El Dorado “Luis Carlos Galán Sarmiento” International Airport in Bogotá, Colombia. Materials and methods: A prospective cohort of 212 workers from the El Dorado airport was designed. The follow-up began in June, 2020. A survey was used to characterize health and work conditions.
Every 21 day, a nasopharyngeal swab was taken to identify the presence of SARS-CoV-2 using RT-PCR. We analyzed the behavior of the cycle threshold (ORF1ab and N genes) according to the day of follow-up. Results: In the first three follow-ups of the cohort, we found an incidence of SARS-CoV-2 infection of 16.51%. The proportion of positive contacts was 14.08%. The median threshold for cycle threshold was 33.53. Conclusion: We characterized the asymptomatic SARS-CoV-2 infection in a cohort of workers. The identification of asymptomatic infected persons continues to be a challenge for epidemiological surveillance systems.
Introducción. La pandemia de COVID-19 ha ocasionado cerca de 25 millones de casos en el mundo. Se ha descrito que los pacientes asintomáticos pueden ser fuentes de transmisión. Sin embargo, es difícil detectarlos y no es claro su papel en la dinámica de transmisión del virus, lo que obstaculiza la implementación de estrategias para la prevención.
Objetivo. Describir el comportamiento de la infección asintomática por SARS-CoV-2 en una cohorte de trabajadores del Aeropuerto Internacional El Dorado “Luis Carlos Galán Sarmiento” de Bogotá, Colombia. Materiales y métodos. Se diseñó una cohorte prospectiva de trabajadores del Aeropuerto El Dorado. El seguimiento se inició en junio de 2020 con una encuesta a cada trabajador para caracterizar sus condiciones de salud y trabajo. Cada 21 días se tomó una muestra de hisopado nasofaríngeo para detectar la presencia del SARS-CoV-2 mediante reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR). Se analizó el comportamiento del umbral del ciclo (cycle threshold) de los genes ORF1ab y N según el día de seguimiento.
Resultados. En los primeros tres seguimientos de la cohorte se encontró una incidencia de la infección por SARS-CoV-2 del 16,51 %. La proporción de contactos positivos fue del 14,08 %. La mediana del umbral del ciclo fue de 33,53. Conclusión. Se determinaron las características de la infección asintomática por el SARSCoV-2 en una cohorte de trabajadores. La detección de infectados asintomáticos sigue siendo un reto para los sistemas de vigilancia epidemiológica.
For use with PE5700, MJ-Opticon & other single color systems, ABI7000, ABI7300, ABI7500, ABI7900, ABI StepOne, StepOne plus, MJ-Opticon2, MJ-chromo4, MX3000P, MX3005P, Smart Cycler II, Rotor-Gene 6000, LightCycler 480, CFX 96, Life 96, Slan 96, iCycl
Description: Novel Coronavirus (2019-nCoV) Real Time RT-PCR Kit is used for the qualitative detection of a novel coronavirus, which was identified in 2019 at Wuhan City, Hubei Province, China, in upper respiratory tract specimens (nasopharyngeal extracts, deep cough sputum, etc.) and lower respiratory tract specimens (alveoli irrigation fluid, etc.) by real time PCR systems.