Ultrasensitive detection and quantification of viral nucleic acids with Raindance droplet digital PCR (ddPCR).
Methods
; 201: 49-64, 2022 05.
Article
in English
| MEDLINE | ID: covidwho-1213579
ABSTRACT
Sensitive detection of viral nucleic acids is critically important for diagnosis and monitoring of the progression of infectious diseases such as those caused by SARS-CoV2, HIV-1, and other viruses. In HIV-1 infection cases, assessing the efficacy of treatment interventions that are superimposed on combination antiretroviral therapy (cART) has benefited tremendously from the development of sensitive HIV-1 DNA and RNA quantitation assays. Simian immunodeficiency virus (SIV) infection of Rhesus macaques is similar in many key aspects to human HIV-1 infection and consequently this non-human primate (NHP) model has and continues to prove instrumental in evaluating HIV prevention, treatment and eradication approaches. Cell and tissue associated HIV-1 viral nucleic acids have been found to serve as useful predictors of disease outcome and indicators of treatment efficacy, highlighting the value of and the need for sensitive detection of viruses in cells/tissues from infected individuals or animal models. However, viral nucleic acid detection and quantitation in such sample sources can often be complicated by high nucleic acid input (that is required to detect ultralow level viruses in, for example, cure research) or inhibitors, leading to reduced detection sensitivity and under-quantification, and confounded result interpretation. Here, we present a step-by-step procedure to quantitatively recover cell/tissue associated viral DNA and RNA, using SIV-infected Rhesus macaque cells and tissues as model systems, and subsequently quantify the viral DNA and RNA with an ultrasensitive SIV droplet digital PCR (ddPCR) assay and reverse transcription ddPCR (RT-ddPCR) assay, respectively, on the Raindance ddPCR platform. The procedure can be readily adapted for a broad range of applications where highly sensitive nucleic acid detection and quantitation are required.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Nucleic Acids
/
HIV Infections
/
Simian Acquired Immunodeficiency Syndrome
/
HIV-1
/
Simian Immunodeficiency Virus
/
COVID-19
Type of study:
Diagnostic study
/
Experimental Studies
/
Prognostic study
Limits:
Animals
Language:
English
Journal:
Methods
Journal subject:
Biochemistry
Year:
2022
Document Type:
Article
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