Application of Synthetic Standard Curves for Absolute Quantification of Hepatitis A and E by Real-Time PCR

Tourinho, Renata Santos; Almeida, Camilla Rodrigues de; Lemos, Andreza Sálvio; Vieira, Yasmine Rangel; Schmidt‐Chanasit, Jonas; Paula, Vanessa Salete de

doi:10.23937/2378-3648/1410013

Cited by 15 publications

(17 citation statements)

References 14 publications

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“…) were used with a synthetic curve for ORF3 detection (Tourinho et al . ) and an internal control. Jothikumar's protocol (Jothikumar et al .…”

Section: Discussionmentioning

confidence: 99%

“…Aiming to develop an efficient methodology, probes and primers described by Jothikumar (Jothikumar et al 2006) were used with a synthetic curve for ORF3 detection (Tourinho et al 2015) and an internal control. Jothikumar's protocol (Jothikumar et al 2006) was used because it is considered to have the best sensibility and specificity according to Mokhtari et al (2013) who compared in-house and commercial methodologies for HEV RNA detection, achieving a detection limit of 250 copies per ml (44Á5 IU ml À1 ).…”

Section: Discussionmentioning

confidence: 99%

“…The TaqMan probe-based quantitative PCR assay (Taq-Man qPCR) described by Jothikumar et al (2006) was improved for quantification using a synthetic DNA standard curve (Tourinho et al 2015) with a nucleic acidbased exogenous internal positive control (IPC) added to the samples before the RT-qPCR reaction (TaqMan, Life Technologies, Carlsbad, CA). Positive faecal and serum samples obtained from pigs were used as positive controls.…”

Section: Methodsmentioning

confidence: 99%

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Detection and quantification of hepatitis E virus in the absence of IgG and IgM anti-HEV in HIV-positive patients

Salvio

Lopes

Almeida³

et al. 2018

J Appl Microbiol

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“…) were used with a synthetic curve for ORF3 detection (Tourinho et al . ) and an internal control. Jothikumar's protocol (Jothikumar et al .…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Detection and quantification of hepatitis E virus in the absence of IgG and IgM anti-HEV in HIV-positive patients

Salvio

Lopes

Almeida³

et al. 2018

J Appl Microbiol

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“…In qPCR, the viral load is measured as the copy number per cell or percentage of total DNA by using a standard curve. A standard curve is generated by qPCR using a dilution series of a DNA template, which is commonly generated from plasmid DNA or DNA oligonucleotides ( Tourinho et al 2015 ). The advantage of using DNA oligonucleotides is that only the nucleotide sequence needs to be synthesized.…”

mentioning

confidence: 99%

“…The advantage of using DNA oligonucleotides is that only the nucleotide sequence needs to be synthesized. For laboratories that do not have enough space or funding for molecular cloning, synthetic curves could be used as an alternative for quantification ( Tourinho et al 2015 ). Based on these advantages, the aim of this study was to evaluate the potential use of a DNA synthetic oligo as a standard for HHV-1 and HHV-2 quantification.…”

mentioning

confidence: 99%

Diagnosis of human herpes virus 1 and 2 (HHV-1 and HHV-2): use of a synthetic standard curve for absolute quantification by real time polymerase chain reaction

Lima

Silva

Schmidt‐Chanasit

et al. 2017

Mem. Inst. Oswaldo Cruz

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The use of quantitative real time polymerase chain reaction (qPCR) for herpesvirus detection has improved the sensitivity and specificity of diagnosis, as it is able to detect shedding episodes in the absence of clinical lesions and diagnose clinical specimens that have low viral loads. With an aim to improve the detection and quantification of herpesvirus by qPCR, synthetic standard curves for human herpesvirus 1 and 2 (HHV-1 and HHV-2) targeting regions gD and gG, respectively, were designed and evaluated. The results show that synthetic curves can replace DNA standard curves in diagnostic herpes qPCR.

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Evaluation of enterovirus concentration, species identification, and cerebrospinal fluid parameters in patients of different ages with aseptic meningitis in São Paulo, Brazil

Honorato,

Ferreira,

Domingues

et al. 2024

Journal of Medical Virology

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Human enteroviruses (EV) are the most common cause of aseptic meningitis worldwide. Data on EV viral load in cerebrospinal fluid (CSF) and related epidemiological studies are scarce in Brazil. This study investigated the influence of EV viral load on CSF parameters, as well as identifying the involved species. CSF samples were collected in 2018–2019 from 140 individuals at The Hospital das Clínicas, São Paulo. The EV viral load was determined using real‐time quantitative polymerase chain reaction, while EV species were identified by 5′UTR region sequencing. Median viral load was 5.72 log10 copies/mL and did not differ by subjects' age and EV species. Pleocytosis was observed in 94.3% of cases, with the highest white blood cell (WBC) counts in younger individuals. Viral load and WBC count were correlated in children (p = 0.0172). Elevated lactate levels were observed in 60% of cases and correlated with the viral load in preteen‐teenagers (p = 0.0120) and adults (p = 0.0184). Most individuals had normal total protein levels (70.7%), with higher in preteen‐teenagers and adults (p < 0.0001). By sequencing, 8.2% were identified as EV species A and 91.8% as species B. Age‐specific variations in CSF characteristics suggest distinct inflammatory responses in each group.

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Application of Synthetic Standard Curves for Absolute Quantification of Hepatitis A and E by Real-Time PCR

Cited by 15 publications

References 14 publications

Detection and quantification of hepatitis E virus in the absence of IgG and IgM anti-HEV in HIV-positive patients

Detection and quantification of hepatitis E virus in the absence of IgG and IgM anti-HEV in HIV-positive patients

Diagnosis of human herpes virus 1 and 2 (HHV-1 and HHV-2): use of a synthetic standard curve for absolute quantification by real time polymerase chain reaction

Evaluation of enterovirus concentration, species identification, and cerebrospinal fluid parameters in patients of different ages with aseptic meningitis in São Paulo, Brazil

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