Single-tube quantitative RT-PCR for monitoring of the response to IFN treatment in CML patients

Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening.

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Direct Quantitation of RNA Transcripts by Competitive Single-Tube RT-PCR and Capillary Electrophoresis

BioTechniques ◽

10.2144/98251rr01 ◽

1998 ◽

Vol 25 (1) ◽

pp. 130-137 ◽

Cited By ~ 26

Author(s):

N.D. Borson ◽

M.A. Strausbauch ◽

P.J. Wettstein ◽

R.P. Oda ◽

S.L. Johnston ◽

...

Keyword(s):

Capillary Electrophoresis ◽

Rt Pcr ◽

Single Tube ◽

Rna Transcripts

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A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

Virology Journal ◽

10.1186/1743-422x-5-94 ◽

2008 ◽

Vol 5 (1) ◽

pp. 94 ◽

Cited By ~ 19

Author(s):

Alexandra Scipioni ◽

Axel Mauroy ◽

Dominique Ziant ◽

Claude Saegerman ◽

Etienne Thiry

Keyword(s):

Sybr Green ◽

Rt Pcr ◽

Pcr Assay ◽

Single Tube ◽

And Control

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Development of Single-tube Multiplex Immunocapture RT-PCR Assay for Simultaneous Detection of Two Pepper Tobamoviruses

The Plant Pathology Journal ◽

10.5423/ppj.2006.22.2.164 ◽

2006 ◽

Vol 22 (2) ◽

pp. 164-167 ◽

Cited By ~ 1

Author(s):

Jae-Hyun Kim ◽

Gug-Seoun Choi ◽

Jeong-Soo Kim ◽

Sin-Ho Lee ◽

Jang-Kyung Choi ◽

...

Keyword(s):

Simultaneous Detection ◽

Rt Pcr ◽

Pcr Assay ◽

Single Tube

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Typing of Dengue Viruses in Clinical Specimens and Mosquitoes by Single-Tube Multiplex Reverse Transcriptase PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.36.9.2634-2639.1998 ◽

1998 ◽

Vol 36 (9) ◽

pp. 2634-2639 ◽

Cited By ~ 142

Author(s):

Eva Harris ◽

T. Guy Roberts ◽

Leila Smith ◽

John Selle ◽

Laura D. Kramer ◽

...

Keyword(s):

Reverse Transcriptase ◽

Dengue Fever ◽

Internal Control ◽

Extraction Procedure ◽

Rna Degradation ◽

Viral Rna ◽

Clinical Samples ◽

Rt Pcr ◽

Single Tube ◽

Dengue Viruses

In recent years, dengue viruses (serotypes 1 to 4) have spread throughout tropical regions worldwide. In many places, multiple dengue virus serotypes are circulating concurrently, which may increase the risk for the more severe form of the disease, dengue hemorrhagic fever. For the control and prevention of dengue fever, it is important to rapidly detect and type the virus in clinical samples and mosquitoes. Assays based on reverse transcriptase (RT) PCR (RT-PCR) amplification of dengue viral RNA can offer a rapid, sensitive, and specific approach to the typing of dengue viruses. We have reduced a two-step nested RT-PCR protocol to a single-tube reaction with sensitivity equivalent to that of the two-step protocol (1 to 50 PFU) in order to maximize simplicity and minimize the risk of sample cross-contamination. This assay was also optimized for use with a thermostable RT-polymerase. We designed a plasmid-based internal control that produces a uniquely sized product and can be used to control for both reverse transcription or amplification steps without the risk of generating false-positive results. This single-tube RT-PCR procedure was used to type dengue viruses during the 1995 and 1997-1998 outbreaks in Nicaragua. In addition, an extraction procedure that permits the sensitive detection of viral RNA in pools of up to 50 mosquitoes without PCR inhibition or RNA degradation was developed. This assay should serve as a practical tool for use in countries where dengue fever is endemic, in conjunction with classical methods for surveillance and epidemiology of dengue viruses.

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Therapeutic effectiveness of interferon-alpha 2b against COVID-19: the Cuban experience

10.1101/2020.05.29.20109199 ◽

2020 ◽

Cited By ~ 3

Author(s):

R Pereda ◽

D González ◽

HB Rivero ◽

JC Rivero ◽

A Pérez ◽

...

Keyword(s):

Case Fatality ◽

Complete Recovery ◽

Treated Group ◽

Preliminary Evidence ◽

Type I Interferons ◽

Therapeutic Effectiveness ◽

Type I ◽

Rt Pcr ◽

Interferon Alpha 2B ◽

Ifn Treatment

ABSTRACTBackgroundEffective therapies are needed to control the SARS-Cov-2 infection pandemic and reduce mortality associated with COVID-19. Several clinical studies have provided evidence for the antiviral effects of type I interferons (IFNs) in patients with respiratory coronaviruses. This study assessed the therapeutic efficacy of IFN-α2b in patients infected with SARS-CoV-2 during the first month after the outbreak began in Cuba.MethodThis multicenter prospective observational study was conducted in 16 hospitals in 8 Cuban provinces. Participants were patients with confirmed SARS-CoV-2 infection detected from throat swab specimens by real time RT-PCR who gave informed consent and had no contraindications for IFN treatment. Patients received therapy as per the Cuban COVID protocol, that included a combination of oral antivirals (lopinavir/ritonavir and chloroquine) with intramuscular administration of IFN-α2b (Heberon® Alpha R, Center for Genetic Engineering and Biotechnology, Havana), 3 times per week, for 2 weeks. The primary endpoint was the proportion of patients discharged from hospital (without clinical and radiological symptoms and non-detectable virus by RT-PCR). The secondary endpoint was the case fatality rate (CFR), defined as the number of confirmed deaths divided by the number of confirmed cases.ResultsFrom March 11th to April 14th, 814 patients were confirmed SARS-CoV-2 positive in Cuba, 761 (93.4%) were treated with Heberon® Alpha R and 53 received the approved protocol without IFN treatment. The proportion of fully recovered patients was higher in the IFN-treated compared with non-IFN treated group (95.4% vs 26.1%, p<0.01). The CFR for all patients was 2.95%, and for those patients who received IFN-α2b the CFR was reduce to 0.92. The estimated global CFR is 6.34% and 4.05% for the Americas reported by WHO and PAHO, respectively. In this study, 82 patients (10.1%) required intensive care and, of these, 42 (5.5%) were treated with IFN.ConclusionsThis report provides preliminary evidence for the therapeutic effectiveness of IFNα-2b for COVID-19 and suggests that the use of Heberon® Alpha R may contribute to complete recovery.

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