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

Abstract

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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