Simultaneous Detection and Differentiation of Human Papillomavirus Genotypes 6, 11, 16 and 18 by AllGlo Quadruplex Quantitative PCR

Yu, Daojun; Chen, Yu; Wu, Shuchao; Wang, Baohong; Tang, Yi‐Wei; Li, Lanjuan

doi:10.1371/journal.pone.0048972

Cited by 17 publications

(17 citation statements)

References 42 publications

(47 reference statements)

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“…AllGlo technology, the latest generation of quantitative fluorescent probes, enables the creation of simple and efficient high-throughput diagnostics with good reproducibility and high sensitivity and specificity (Yu et al, 2012). This technology has been widely used for agricultural and medical studies, e.g.…”

Section: Discussionmentioning

confidence: 99%

“…With permission from the outpatients and inpatients, 300 clinical specimens, including 130 sputum specimens and 170 nasopharyngeal swab specimens, were collected from Hangzhou First People's Hospital, China. According to Yu et al (2012), DNA was extracted from all samples (details are given in S1.1, available in the online Supplementary Material).…”

Section: Methodsmentioning

confidence: 99%

“…The indicators of the quadruplex qPCR noted by Yu et al (2012), including sensitivity, specificity, repeatability and standard curves, were analysed (details in S1.4).…”

Section: Allglo Multiplex Real-time Qpcrmentioning

confidence: 99%

“…Nevertheless, in current reports of qPCR methods three pathogens (MP, EBV and HCMV) and an internal control in a single tube have not yet been simultaneously detected. In this study, novel AllGlo probes (Alle Logic Biosciences), which yield higher sensitivity and specificity than traditional TaqMan probes (Bai et al, 2014;Yu et al, 2012;Zhang et al, 2014), were designed and used to develop a multiplex qPCR method that rapidly, reliably and simultaneously detected and identified the three pathogens in a single tube in clinical samples. Importantly, we introduced a human housekeeping gene, GAPDH, as an internal control to strengthen the surveillance and control of detection quality.…”

Section: Introductionmentioning

confidence: 99%

“…This difference was probably due to competitive effects of increasing the amount of nucleic acid templates, whether capable of participating in qPCR or not, which indicated the quadruplex qPCR reaction system with a mixed plasmid template might exhibit mutual interference among the primers and probes. However, the difference of one C t value did not affect the sensitivity of AllGlo quadruplex qPCR and it was considered acceptable in daily clinical practice.AllGlo technology, the latest generation of quantitative fluorescent probes, enables the creation of simple and efficient high-throughput diagnostics with good reproducibility and high sensitivity and specificity (Yu et al, 2012). This technology has been widely used for agricultural and medical studies, e.g.…”

mentioning

confidence: 99%

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Rapid and combined detection of Mycoplasma pneumoniae, Epstein–Barr virus and human cytomegalovirus using AllGlo quadruplex quantitative PCR

Chen

Pan

et al. 2016

Journal of Medical Microbiology

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Acute respiratory infections (ARIs) cause substantial morbidity and mortality worldwide. The causes of ARI are dynamic, and co-infections of Mycoplasma pneumoniae, Epstein-Barr virus and human cytomegalovirus are recently developed causes of ARI. Here, we established a quadruplex quantitative PCR (qPCR) method to rapidly identify and simultaneously detect a single infection or co-infection of these three pathogens and an internal control in a single tube using AllGlo probes. The analysis demonstrated a wide linear range of detection from 10 1 to 10 8 copies per test and a low coefficient of variation of less than 5 %. The amplification efficiencies were all close to 1, and the correlation coefficients (r 2 ) were all greater than 0.99. We found no significant difference in a comparative reagent test (P>0.05). Moreover, the results of tests on clinical samples using AllGlo quadruplex qPCR and TaqMan uniplex qPCR were in near-perfect agreement (k =0.97). Clinically, the availability of this method will enable better differential diagnosis, disease surveillance and controlled outcomes. INTRODUCTIONAcute respiratory infections (ARIs) are a common cause of morbidity and mortality among adults and children, and the aetiology of ARI is dynamically changing (Verani et al., 2013; Zumla et al., 2014). During the past decade, studies have demonstrated that mycoplasma and viral infections are increasing (Jain et al., 2015;Rasmussen et al., 2010;Wallihan & Ramilo, 2014). Mycoplasma pneumoniae (MP), Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) are common pathogens of respiratory infections . EBV and HCMV frequently cause infectious mononucleosis (IM) accompanied by respiratory tract infections (Grilli et al., 2012;Restrepo-Gualteros et al., 2014;Wang et al., 2010;Xiong et al., 2014). In contrast, MP is characterized by acute onset, rapid progress and long course (Meyer Sauteur et al., 2014;Waites & Talkington, 2004). Studies have indicated that coinfections between MP, EBV or HCMV can lead to severe clinical consequences, so that there is a particular need to vigilantly monitor IM-like symptoms (Huijskens et al., 2014;Ito et al., 2009;Klein et al., 2013;Li et al., 2014;Meyer Sauteur et al., 2014;Narita, 2010; Schneider et al., 2013;Wang et al., 2010). Because co-infection often cannot be excluded, singlepathogen detection is likely to cause misdiagnoses and missed diagnoses. Therefore, a reliable, sensitive and rapid test that can simultaneously detect and differentiate among MP, EBV and HCMV should be established to aid in diagnosis.Clinically, a correct aetiological diagnosis of MP, EBV or HCMV infection relies heavily on laboratory examination, including microbiological culture, serology, quantitative PCR (qPCR) and so on (Cannon et al., 2010;Daxboeck et al., 2003;Reddington et al., 2013). To our knowledge, the methods based on serology and qPCR are widely used to detect these pathogens at present. However, owing to their high sensitivity, specificity and throughput, qPCR-based assays are increasingly being a...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The indicators of the quadruplex qPCR noted by Yu et al (2012), including sensitivity, specificity, repeatability and standard curves, were analysed (details in S1.4).…”

Section: Allglo Multiplex Real-time Qpcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Rapid and combined detection of Mycoplasma pneumoniae, Epstein–Barr virus and human cytomegalovirus using AllGlo quadruplex quantitative PCR

Chen

Pan

et al. 2016

Journal of Medical Microbiology

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Human Papillomaviruses

Babady

2016

Manual of Commercial Methods in Clinical Microbiology

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Reliable detection of Human papillomavirus in recurrent laryngeal papillomatosis and associated carcinoma of archival tissue

2015

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Recurrent laryngeal papillomatosis (RLP) is, although benign, a challenging disease for both, the patient and the treating physician. Maximum disease control with minimum intervention is considered to be the gold standard. However, patients have to undergo repeating surgical interventions. Human papillomavirus (HPV), mainly so called low risk types, are thought to be responsible for the development of RLP. But, there is still some controversy over the true prevalence of HPV and the virus-specific molecular diagnostic of choice. Therefore archival tissue samples from 44 patients with RLP at laryngeal site, out of which eight developed laryngeal cancer, was screened for presence of HPV through various molecular approaches. Results from these different methodologies were compared between each other and with patient's characteristics. The overall detection rates of HPV with the various methods used in this study were: HPV16 E6/E7 PCR: 0%; GP5+/6+ PCR: 4.5%; CDKN2A/p16 immunohistochemistry: 6.8%; in-situ hybridization for low and high risk HPV types: 52.3%; HPV6/11 L1 PCR: 72.7% and HPV6/11 E6 PCR: 79.5%. Disease progression showed no apparent dependence of the detected HPV type or clinical variables like age at diagnosis, sex, or additional drug application (Cidofovir and Bevacizumab). In conclusion, the broad-spectrum PCRs alone or in combination with immunohistochemistry of CDKN2A/p16 and in-situ hybridization are unsuitable for HPV detection in RLP. Based on the findings presented in this study the type specific PCRs targeting the E6 open reading frame are clearly superior in detection of HPV in this tumor entity.

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Simultaneous Detection and Differentiation of Human Papillomavirus Genotypes 6, 11, 16 and 18 by AllGlo Quadruplex Quantitative PCR

Cited by 17 publications

References 42 publications

Rapid and combined detection of Mycoplasma pneumoniae, Epstein–Barr virus and human cytomegalovirus using AllGlo quadruplex quantitative PCR

Rapid and combined detection of Mycoplasma pneumoniae, Epstein–Barr virus and human cytomegalovirus using AllGlo quadruplex quantitative PCR

Human Papillomaviruses

Reliable detection of Human papillomavirus in recurrent laryngeal papillomatosis and associated carcinoma of archival tissue

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