Comparison data of a two-target real-time PCR assay with and without an internal control in detecting Salmonella enterica from cattle lymph nodes

Abstract Background: The orthopox viruses that are pathogenic for humans include variola major virus (VAR), monkeypox virus (MPV), cowpox virus (CPV), and to a lesser extent, camelpox virus (CML) and vaccinia virus (VAC). PCR is a powerful tool to detect and differentiate orthopox viruses, and real-time PCR has the further advantages of rapid turnaround time, low risk of contamination, capability of strain differentiation, and use of multiplexed probes. Methods: We used real-time PCR with fluorescence resonance energy transfer technology to simultaneously detect and differentiate VAR, MPV, CPV/VAC, and CML. An internal control generated by cloning and mutating the PCR target gene facilitated monitoring of PCR inhibition in each individual test reaction. Results: Strain differentiation results showed little interassay variability (CV, 0.4–0.6%), and the test was 100-fold more sensitive than virus culture on Vero cells. Low copy numbers of DNA could be detected with ≥95% probability (235–849 genome copies/mL of plasma). Conclusions: The real-time PCR assay can detect and differentiate human pathogenic orthopox viruses. The use of an internal control qualifies the assay for high sample throughput, as is likely to be needed in situations of suspected acts of biological terrorism, e.g., use of VAR.

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Quantitative monitoring of HCMV DNAlactia in human milk by real time PCR assay: Implementation of internal control contributes to standardization and quality control

Journal of Virological Methods ◽

10.1016/j.jviromet.2016.08.020 ◽

2016 ◽

Vol 237 ◽

pp. 101-106 ◽

Cited By ~ 3

Author(s):

Steffen Hartleif ◽

Katharina Göhring ◽

Rangmar Goelz ◽

Gerhard Jahn ◽

Klaus Hamprecht

Keyword(s):

Quality Control ◽

Human Milk ◽

Real Time ◽

Internal Control ◽

Real Time Pcr ◽

Pcr Assay ◽

Quantitative Monitoring

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PCR and culture for diagnosis of Acanthamoeba keratitis

British Journal of Ophthalmology ◽

10.1136/bjophthalmol-2020-316730 ◽

2020 ◽

pp. bjophthalmol-2020-316730

Author(s):

Helene Yera ◽

Vichita Ok ◽

Fiona Lee Koy Kuet ◽

Naima Dahane ◽

Frédéric Ariey ◽

...

Keyword(s):

Real Time ◽

Internal Control ◽

Real Time Pcr ◽

Latent Class ◽

Rrna Gene ◽

Target Sequence ◽

Small Subunit Rrna ◽

Pcr Assay ◽

Corneal Scraping ◽

Pcr Assays

Background/AimsAcanthamoeba keratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis thus far. In this study, we prospectively compared the performance of five PCR assays on corneal samples for the diagnosis of AK.Methods1217 corneal scraping samples were obtained from patients, for whom an AK was suspected. Sample processing involved both molecular diagnostics and culture. Acanthamoeba PCR assays detected different regions of the Acanthamoeba nuclear small-subunit rRNA gene: three final point PCR assays using Nelson, ACARNA and JDP1–JDP2 pairs of primers, and two real-time PCR assays using Acant primer-probe. Human DNA and internal control were co-amplified in the real-time PCR assay to ensure scraping quality and the absence of inhibitors. In the absence of a gold standard, the performance of each test was evaluated using latent class analysis. Genotypes of Acanthamoeba isolates were also characterised.ResultsEstimated prevalence of AK was 1.32%. The sensitivity of Acanthamoeba diagnostic PCRs (73.3% to 86.7%) did not differ significantly from that of culture (66.7%), or according to the target sequence or the technology. Sensitivity could be increased to 93.8% or 100% by combining two or three assays, respectively. PCR specificity (99.3% to 100%) differed between the assays. T4 was the predominant Acanthamoeba genotype (84.6%).ConclusionsCulture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.

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