Multiplex real‐time PCR for the simultaneous detection of herpes simplex virus, human herpesvirus 6, and human herpesvirus 7

Wada, Kaoru; Mizoguchi, Sachiko; Ito, Yoshinori; Kawada, Junji; Yamauchi, Yohei; Morishima, Tsuneo; Nishiyama, Yukihiro; Kimura, Hiroshi

doi:10.1111/j.1348-0421.2008.00090.x

Cited by 44 publications

(34 citation statements)

References 45 publications

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“…Similarities in standard curves generated by singleplex and multiplex assay quantification across a dynamic range of template concentrations have been previously used to validate multiplex assays (Wei et al, 2008;Shum and Paul, 2009;Wada et al, 2009). Increases in Ct for standards quantified in multiplex assay compared with singleplex assays have been observed previously without negative impact on assay efficiency (Rao et al, 2009;Shum and Paul, 2009;Wada et al, 2009).…”

Section: Utr Products Ofmentioning

confidence: 94%

“…and Paul, 2009). Compared with conventional real-time PCR assays, multiplex PCR assays are more difficult to design due to the potential competition and similarities between primer-probe combinations, annealing temperatures, and effects of multiple primer-probe concentrations (King et al, 2008;Shum and Paul, 2009;Wada et al, 2009). Multiplex assays have been developed to detect bacteria, viruses, parasites, and retroviral vector promoters (Cho et al, 2009;Rao et al, 2009;Veron et al, 2009).…”

Section: Utr Products Ofmentioning

confidence: 99%

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Bovine pyruvate carboxylase 5′ untranslated region variant expression during transition to lactation and feed restriction in dairy cows1

White

Koser

Donkin

2011

Journal of Animal Science

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Pyruvate carboxylase (PC; EC 6.4.1.1) is a critical enzyme for gluconeogenesis and maintenance of tricarboxylic acid (TCA) cycle intermediates, and expression of PC mRNA is increased at calving and during feed restriction. The bovine PC gene contains 3 promoters (3, 2, and 1 from 5' to 3') that produce 6 mRNA 5' variants (A through F). Products of promoter 1, untranslated region (UTR) variants A, B, C, and F are specifically expressed in glucogenic and lipogenic tissues. The objective of this study was to develop a quantitative PCR-based assay for bovine PC 5' UTR variants that would permit simultaneous characterization of PC variant expression and to determine the pattern of PC variant expression during the transition to lactation and during feed restriction. Primer combinations specific to the coding region of PC and the 5' UTR for variants D, E, and F were used with Taqman probes in a real-time PCR multiplex assay to simultaneously determine total PC mRNA and expression of each UTR variant. The intraassay and interassay CV were less than 2 and 10%, respectively. Total PC mRNA and PC 5' UTR variant profile was determined for liver biopsy samples collected from Holstein cows (n = 8) at -28, +1, and +28 d relative to calving (DRTC) and from mid-lactation Holstein cows subjected to either feed restriction (n = 8) or fed for ad libitum intake (n = 8). The expression of PC mRNA corresponding to the coding region of PC and PC 5' UTR variant regions A, B, C, and F increased (P < 0.05) 4-fold with feed restriction and 6-fold at calving. Nuclei isolated from liver biopsy samples and used to determine the rates of PC gene transcription indicate changes in the abundance of PC 5' mRNA variants A, B, C, and F that are due to corresponding changes in the rate of transcription of the bovine PC gene. The data support the use of the multiplex assay described here as a proxy measure of the activity of bovine PC promoter 1. The increased PC mRNA expression observed at calving and during feed restriction is the result of specific increases in 5' UTR variants A, B, C, and F due to increased transcriptional activity of promoter 1 of the PC gene.

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Section: Utr Products Ofmentioning

confidence: 94%

Section: Utr Products Ofmentioning

confidence: 99%

Bovine pyruvate carboxylase 5′ untranslated region variant expression during transition to lactation and feed restriction in dairy cows1

White

Koser

Donkin

2011

Journal of Animal Science

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“…This failure can be explained by the inconsistency between the small volume of CSF available and the large number of viruses potentially responsible for meningitis or encephalitis (15). In the first step, the use of a multiplex PCR approach allowed us to broaden the detection of neurotropic viruses (2,6,7,8,22,29,31,32,41,44). Nevertheless, some multiplex PCR requires confirmation by hybridization of the amplicons to specific probes fixed onto mi- (6).…”

Section: Discussionmentioning

confidence: 99%

Rapid Virological Diagnosis of Central Nervous System Infections by Use of a Multiplex Reverse Transcription-PCR DNA Microarray

Lévêque¹,

Haecke²,

Renois³

et al. 2011

J Clin Microbiol

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“…Previously, we developed multiplex real-time PCR systems that can quantify three viruses simultaneously (20,21) and put the technique into clinical practice after stem cell transplantation. The use of these systems reduces significantly the time and labor required for the examination of each virus.…”

mentioning

confidence: 99%

Multiplex Real-Time PCR Assay for Simultaneous Quantification of BK Polyomavirus, JC Polyomavirus, and Adenovirus DNA

Funahashi¹,

Iwata

Ito

et al. 2010

J Clin Microbiol

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In recent years, virus-induced nephropathy caused mainly by BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), and adenovirus has emerged as a problem in renal transplant patients. In the present study, we developed a multiplex real-time PCR assay to quantify the viral load of BKPyV, JCPyV, and adenovirus simultaneously. The dynamic range covered at least 6 orders of magnitude. This system was specific and reproducible, even in the presence of large amounts of DNA of other viruses. To validate this assay, urine samples from 124 renal transplant patients and serum samples from 18 hemorrhagic cystitis patients after hematopoietic stem cell transplantation were examined. In the urine samples from renal transplant patients, BKPyV was detected in 28 patients (22.6%), JCPyV was detected in 51 patients (41.1%), and adenovirus was detected in 2 patients (1.6%). The maximum amounts of each virus detected were 2.7 ؋ 10 9 , 8.7 ؋ 10 8 , and 1.2 ؋ 10 2 copies/ml, respectively. Decoy cells were observed in 31 patients. The quantities of both BKPyV and JCPyV DNA were greater in samples with decoy cells. Two patients whose BKPyV viral loads exceeded 10 8 copies/ml had elevated serum creatinine levels and were diagnosed with BKPyV nephropathy based on graft biopsies. In serum samples from hemorrhagic cystitis patients, BKPyV, JCPyV, and adenovirus was detected in six, two, and three patients, respectively. Strong correlations were observed between the viral DNA copy numbers determined in the multiplex assays and those determined in single assays. Since this new assay is rapid, sensitive, and specific, it can be used for quantitative analyses of viruses in urine and serum samples after transplantation.Although immunological rejection after renal transplantation has decreased with advances in immunosuppressive therapy, virus-induced nephropathy, which was rare with conventional immunosuppressants, has become a clinical problem. BK polyomavirus (BKPyV) is a prevalent pathogen causing virus-induced nephropathy, and other viruses, including JC polyomavirus (JCPyV) and adenovirus, can cause nephropathy (1, 4, 13). These viruses commonly infect a large number of people in childhood and remain latent in the urinary tract after primary infection. Reactivation with asymptomatic viruria may occur in both immunocompetent subjects and immunocompromised patients.For the early diagnosis and treatment of BKPyV nephropathy, urine cytology is useful for screening high-risk patients (1). The virus-infected urothelial cells called "decoy cells," identified by their typical ground glass intranuclear inclusions on cellular smears stained by the Papanicolaou method, are observed in most patients with BKPyV nephropathy. However, decoy cells are not specific for the presence of BKPyV in urine and can be found in JCPyV and adenovirus infection (1,4,22). Therefore, the detection of viral DNA is essential for a precise diagnosis and the identification of patients at high risk for nephropathy. In addition, hemorrhagic cystitis can be observed after hematopo...

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Multiplex real‐time PCR for the simultaneous detection of herpes simplex virus, human herpesvirus 6, and human herpesvirus 7

Cited by 44 publications

References 45 publications

Bovine pyruvate carboxylase 5′ untranslated region variant expression during transition to lactation and feed restriction in dairy cows1

Bovine pyruvate carboxylase 5′ untranslated region variant expression during transition to lactation and feed restriction in dairy cows1

Rapid Virological Diagnosis of Central Nervous System Infections by Use of a Multiplex Reverse Transcription-PCR DNA Microarray

Multiplex Real-Time PCR Assay for Simultaneous Quantification of BK Polyomavirus, JC Polyomavirus, and Adenovirus DNA

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