Highly sensitive and specific detection of the SARS-CoV-2 Delta variant by double-mismatch allele-specific real time reverse transcription PCR.

Background: The highly transmissible Delta variant of SARS-CoV-2 (B.1.617.

A HML6 endogenous retrovirus on chromosome 3 is upregulated in amyotrophic lateral sclerosis motor cortex.

There is increasing evidence that endogenous retroviruses (ERVs) play a significant role in central nervous system diseases, including amyotrophic lateral sclerosis (ALS). Studies of ALS have consistently identified retroviral enzyme reverse transcriptase activity in patients. Evidence indicates that ERVs are the cause of reverse transcriptase activity in ALS, but it is currently unclear whether this is due to a specific ERV locus or a family of ERVs.

Cautionary Note on Contamination of Reagents Used for Molecular Detection of SARS-CoV-2.

Reverse transcription (RT)-PCR, the principal diagnostic method applied in the world-wide struggle against COVID-19, is capable of detecting a single molecule of a viral genome. Correctly designed and practiced RT-PCR assays for SARS-CoV-2 should not cross react with similar but distinct viral pathogens, such as the coronaviruses associated with the common cold, and should perform with very high analytical sensitivity. This analytical performance is predicated on the ability of the method to detect the presence of the selected nucleic acid target, without detection of a false positive signal.

Quantitative analysis of human endogenous retrovirus-K transcripts in postmortem premotor cortex fails to confirm elevated expression of HERV-K RNA in amyotrophic lateral sclerosis.

Over the past two decades a number of studies have demonstrated activity of the retroviral enzyme reverse transcriptase in the serum of patients with sporadic amyotrophic lateral sclerosis (ALS). Known human exogenous retroviruses such as HIV-1 have been eliminated as possible sources of this activity and investigators have therefore considered the possibility that human endogenous retroviruses (HERVs) might be involved. HERV-K (HML-2) is the most recent retroviral candidate to be proposed following the observation of elevated HERV-K expression in cortical and spinal neurons of ALS patients and the demonstration of HERV-K envelope protein neurotoxicity in vitro and in transgenic mice.

Minor groove binder modification of widely used TaqMan hydrolysis probe for detection of dengue virus reduces risk of false-negative real-time PCR results for serotype 4.

Dengue is a vector-transmitted viral infection that is a significant cause of morbidity and mortality in humans worldwide, with over 50 million apparent cases and a fatality rate of 2.5 % of 0.5 million severe cases per annum in recent years.

Development of an improved RT-qPCR Assay for detection of Japanese encephalitis virus (JEV) RNA including a systematic review and comprehensive comparison with published methods.

Background: Japanese encephalitis virus (JEV) is a major cause of encephalitis in Asia, and the commonest cause of mosquito-borne encephalitis worldwide. Detection of JEV RNA remains challenging due to the characteristic brief and low viraemia, with 0-25% of patients positive, and the mainstay of diagnosis remains detection of anti-JEV IgM antibody.

Methods: We performed a systematic review of published RT-PCR protocols, and evaluated them in silico and in vitro alongside new primers and probes designed using a multiple genome alignment of all JEV strains >9,000nt from GenBank, downloaded from the NCBI website (November 2016).

Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR.

Establishing a cure for HIV is hindered by the persistence of latently infected cells which constitute the viral reservoir. Real-time qPCR, used for quantification of this reservoir by measuring HIV DNA, requires external calibration; a common choice of calibrator is the 8E5 cell line, which is assumed to be stable and to contain one HIV provirus per cell. In contrast, digital PCR requires no external calibration and potentially provides 'absolute' quantification.

Digital PCR dynamic range is approaching that of real-time quantitative PCR.

Digital PCR (dPCR) has been reported to be more precise and sensitive than real-time quantitative PCR (qPCR) in a variety of models and applications. However, in the majority of commercially available dPCR platforms, the dynamic range is dependent on the number of partitions analysed and so is typically limited to four orders of magnitude; reduced compared with the typical seven orders achievable by qPCR. Using two different biological models (HIV DNA analysis and genotyping), we have demonstrated that the RainDrop Digital PCR System (RainDance Technologies) is capable of performing accurate and precise quantification over six orders of magnitude thereby approaching that achievable by qPCR.

Hepatitis C virus quasispecies and pseudotype analysis from acute infection to chronicity in HIV-1 co-infected individuals.

HIV-1 infected patients who acquire HCV infection have higher rates of chronicity and liver disease progression than patients with HCV mono-infection. Understanding early events in this pathogenic process is important. We applied single genome sequencing of the E1 to NS3 regions and viral pseudotype neutralization assays to explore the consequences of viral quasispecies evolution from pre-seroconversion to chronicity in four co-infected individuals (mean follow up 566 days).

Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples.

Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.

Diagnosis of neuroinvasive astrovirus infection in an immunocompromised adult with encephalitis by unbiased next-generation sequencing.

Metagenomic next-generation sequencing (NGS) was used to diagnose an unusual and fatal case of progressive encephalitis in an immunocompromised adult presenting at disease onset as bilateral hearing loss. The sequencing and confirmatory studies revealed neuroinvasive infection of the brain by an astrovirus belonging to a recently discovered VA/HMO clade.

Comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus.

Performing nucleic acid amplification techniques (NAATs) in digital format using limiting dilution provides potential advantages that have recently been demonstrated with digital polymerase chain reaction (dPCR). Key benefits that have been claimed are the ability to quantify nucleic acids without the need of an external calibrator and a greater resistance to inhibitors than real-time quantitative PCR (qPCR). In this study, we evaluated the performance of four NAATs, qPCR, dPCR, real-time quantitative loop mediated isothermal amplification (qLAMP), and digital LAMP (dLAMP), for the detection and quantification of human cytomegalovirus (hCMV).

Evaluation of an ethidium monoazide-enhanced 16S rDNA real-time polymerase chain reaction assay for bacterial screening of platelet concentrates and comparison with automated culture.

Background: Culture-based systems are currently the preferred means for bacterial screening of platelet (PLT) concentrates. Alternative bacterial detection techniques based on nucleic acid amplification have also been developed but these have yet to be fully evaluated. In this study we evaluate a novel 16S rDNA polymerase chain reaction (PCR) assay and compare its performance with automated culture.

The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments.

There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR).

Minor groove binder modification of widely used TaqMan probe for hepatitis E virus reduces risk of false negative real-time PCR results.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in many parts of the developing world. It is responsible for both sporadic infections and large scale epidemics and may be associated with significant mortality during pregnancy. Over the past two decades many serological and nucleic acid based diagnostic tests for HEV have been developed, including several reverse transcription real-time polymerase chain reaction assays (RT-qPCR).

Development of an ethidium monoazide-enhanced internally controlled universal 16S rDNA real-time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates.

Background: Bacterial contamination of platelet (PLT) concentrates remains a problem for blood transfusion services. Culture-based bacterial screening techniques are available but offer inadequate speed and sensitivity. Alternative techniques based on polymerase chain reaction (PCR) amplification have been described but their performance is often compromised by traces of bacterial DNA in reagents.

No evidence of XMRV or related retroviruses in a London HIV-1-positive patient cohort.

Background: Several studies have implicated a recently discovered gammaretrovirus, XMRV (Xenotropic murine leukaemia virus-related virus), in chronic fatigue syndrome and prostate cancer, though whether as causative agent or opportunistic infection is unclear. It has also been suggested that the virus can be found circulating amongst the general population. The discovery has been controversial, with conflicting results from attempts to reproduce the original studies.

Analysis of XMRV integration sites from human prostate cancer tissues suggests PCR contamination rather than genuine human infection.

XMRV is a gammaretrovirus associated in some studies with human prostate cancer and chronic fatigue syndrome. Central to the hypothesis of XMRV as a human pathogen is the description of integration sites in DNA from prostate tumour tissues. Here we demonstrate that 2 of 14 patient-derived sites are identical to sites cloned in the same laboratory from experimentally infected DU145 cells.