Development of a novel internal control for a real-time PCR for HSV DNA types 1 and 2

Hodgson, Julian; Zuckerman, Mark; Smith, M. A.

doi:10.1016/j.jcv.2006.12.014

Cited by 10 publications

(6 citation statements)

References 21 publications

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“…Of note, this control is absent in the HSV test routinely used in the laboratory. As described with other HSV molecular assays (36)(37)(38)(39), the prevalence of PCR inhibition was low in this study (1.1%) and concerned CSF specimens only. Approximately 7% of the tested CSF samples were found invalid; the test would need to be validated on a larger number of CSF samples for verifying whether this observation is anecdotal or corresponds to a recurrent phenomenon with this type of sample, which could be a limitation for the test.…”

Section: Discussionsupporting

confidence: 56%

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens

et al. 2015

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A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV)DNAH erpes simplex viruses 1 and 2 (HSV-1 and -2, respectively) and varicella-zoster virus (VZV) are human double-stranded DNA viruses belonging to the Herpesviridae family. In immunocompetent hosts, benign vesicular eruption of the skin and mucosa occurs during either the primary infection or reactivation episodes (1). However, life-threatening manifestations, mainly related to virus neurovirulence, can be observed (2), HSV-1 and VZV being the first and second most common causes of viral encephalitis, respectively (3, 4). These viruses are associated with mortality and morbidity, especially when treatment with acyclovir is delayed (5). HSV-2 is more likely responsible for meningitis (1). HSV-2 and VZV are also implicated in neonatal infections (1,6,7).Due to similarities in the clinical presentations of the diseases caused by these 3 herpesviruses and other pathogens, virological testing is often needed. Accurate and specific diagnosis is of special importance in cases of severe infection or in infection occurring in immunosuppressed patients who can present atypical clinical pictures (8). In replacement of cell culture and immune detection of viral antigens, molecular assays are currently the main tool used for the virological diagnosis of infections related to HSV and VZV. This trend is due to the multiple advantages of nucleic acid testing (NAT), including the rapid availability of results, its adaptation to a wide diversity of clinical specimens, the capacity for automation, and, mostly, its high sensitivity and specificity. Several FDA-approved or CE-marked in vitro diagnostic (IVD) kits are available for detecting these viruses in various specimens (9-11). However, all of them propose separate detection for HSV and VZV DNA, and only a few multiplex assays including both agents have been published as laboratory-developed tests (LDTs) (10, 12).Although detection of viral DNA is the gold standard for the virological diagnosis of neurological severe infections (3), to date, no commercial fully automated PCR assay detecting both HSV and VZV is available. However, an FDA-approved assay for HSV detection in cerebrospinal fluid (CSF) was recently validated for clinical use (13,14). As low viral loads (Ͻ500 copies/ml) are not infrequent in CSF of patients suffering encephalitis associated with , it was recommended to reach a sensitivity of at least 200 copies/ml for the detection of this agent in CSF (21). The same need for sensitive tests is found with encephalitis associated with VZV (15, 22). The recently commercialized BD Max system (Becton Dickinson, Pont-de-Claix, France) combines the extraction and amplification steps on the same instrument, shortening the hands-on time and allowing its use in emergency molecular diagnostic testing. Dedicated analyte kits (23-26) but also master mix from other companies (27) and laboratory-developed methods (28) can be

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Section: Discussionsupporting

confidence: 56%

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens

et al. 2015

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“…The internal control was derived from a plasmid construct consisting of a 147 bp fragment of Promega's pGEM-T (Promega UK Ltd, Southampton, UK) vector with the VZV forward and reverse primer sequences incorporated into the 5′ and 3′ ends forming a chimeric molecule, amplified by the VZV primers and detected by a sequence-specific TaqMan probe (Applied Biosystems, Warrington, UK), based on the original method described previously 13. To produce the RNA internal control, the fragment was cloned back into the pGEM-T vector, reverse transcribed with Promega's Riboprobe in vitro Transcription System, as described by the manufacturer, isolated, purified and quantified using standard molecular biology techniques.…”

Section: Methodsmentioning

confidence: 99%

Lung function prior to viral lower respiratory tract infections in prematurely born infants

Drysdale

Wilson

Alcazar-Paris

et al. 2011

Thorax

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Objective Prematurely born infants who develop respiratory syncytial virus (RSV) lower respiratory tract infections (LRTIs) have lung function abnormalities at follow-up. The aim of this study was to determine whether prematurely born infants who developed symptomatic RSV, or other viral LRTI(s), had poorer premorbid lung function than infants who did not develop LRTIs during the RSV season. Methods Lung function (functional residual capacity (FRC), compliance (Crs) and resistance (Rrs) of the respiratory system) was measured at 36 weeks postmenstrual age. After neonatal unit discharge, nasopharyngeal aspirates were obtained whenever the infants had an LRTI, regardless of whether this was in the community or in hospital. Nasopharyngeal aspirates were examined for RSV A and B, rhinovirus, influenza A and B, parainfluenza 1, 2 and 3, human metapneumovirus and adenovirus. Results 159 infants with a median gestational age of 34 (range 23e36) weeks were prospectively followed. 73 infants developed LRTIs: 27 had at least one RSV LRTI and 31 had at least one other viral LRTI, but not an RSV LRTI. Overall, there were no significant differences in the FRC (p¼0.54), Crs (p¼0.11) or Rrs (p¼0.12) results between those who developed an RSV or other viral LRTI and those who did not develop an LRTI. Infants with RSV or other viral LRTIs who were admitted to hospital compared with those who were not had higher Rrs results (p¼0.033 and p¼0.039, respectively). Conclusion Diminished premorbid lung function may predispose prematurely born infants to severe viral LRTIs in infancy.

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“…Using the last strategy would add an extra step to the detection because following the PCR; the PCR product should be subjected to enzyme digestion. Occasionally, false negative results may occur due to fail of enzymatic digestions (22-28). Hodgson and his colleagues (2006) developed a competitive IPC for detection of HSV types 1 and HSV 2.…”

Section: Discussionmentioning

confidence: 99%

“…Hodgson and his colleagues (2006) developed a competitive IPC for detection of HSV types 1 and HSV 2. They made a DNA fragment containing a primer annealing site for target gene with the heterologous region at the middle (derived from the pGEM plasmid) (22). …”

Section: Discussionmentioning

confidence: 99%

Construction and Evaluation of a Novel Internal Positive Control (IPC) for Detection of Coxiella burnetii by PCR

Majidzadeh

Mohseni

Soleimani

2014

Jundishapur J Microbiol

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Background:Due to the limitations of the classical methods to detect Coxiella burnetii, direct diagnosis of the pathogen using PCR techniques is still the preferable approach. However, false negative results owing to the presence of PCR inhibitors are troublesome.Objectives:In order to identify the inhibitors during PCR assay, an internal positive control (IPC) was designed based on 16SrRNA gene of C. burnetii.Materials and Methods:In the current study, the initial and ending parts of the target gene in an external positive control plasmid (pTZ57R/T-16S) amplified using internal primers which had a BglII restriction site on the 5´ends. Both PCR products (fragments 1 and 2) were cloned into pTZ57R/T vector. Following BglII enzyme digestion, the two obtained linear plasmids were ligated. The ligation product was transformed into Escherichia coli Top10 Fʹ. Screening of the desired recombinant clone was carried out using colony PCR.Results:The size of the PCR product was equal to the sum of the first and second fragments. Sequencing confirmed the presence of the desire insert (IPC sequence) in recombinant plasmid. Consequently, the IPC fragment was longer than the target gene while both ends had similar attachments to the same primer pair.Conclusions:The results showed that direct fusion of the recombinant plasmids containing the initial and ending parts of the target gene are simple and cost-effective techniques for increasing the length of the fragment and constructing IPC.

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Development of a novel internal control for a real-time PCR for HSV DNA types 1 and 2

Cited by 10 publications

References 21 publications

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens

Lung function prior to viral lower respiratory tract infections in prematurely born infants

Construction and Evaluation of a Novel Internal Positive Control (IPC) for Detection of Coxiella burnetii by PCR

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