A low density oligonucleotide microarray for the detection of viral and atypical bacterial respiratory pathogens

Abstract

Acute respiratory tract infections are a major cause of morbidity and mortality worldwide and exert a considerable economic burden on healthcare systems. Acute respiratory tract infections of the upper and lower respiratory tract are caused by a wide variety of viral and bacterial pathogens, which require comprehensive laboratory investigations. Conventional serological and immunofluorescence-based diagnostic methods for acute respiratory tract infections lack sensitivity when compared to polymerase chain reaction (PCR)-based approaches and the development of new diagnostic methodologies is required, to provide accurate, sensitive and rapid diagnoses. In the present study, a PCR-based low density oligonucleotide microarray was developed for the detection of 16 viral and two atypical bacterial pathogens. The performance of this DNA microarray-based analysis exhibited comparable sensitivities and specificities to multiplex real-time reverse transcription polymerase chain reactions (rtPCRs) confirming the potential diagnostic utility of the method. In contrast to routine multiplex PCR, the microarray incorporates an intrinsic redundancy as multiple and non-identical probes per target on the array allow direct intra-assay confirmation of positives. This study demonstrates that microarray technology provides a viable alternative to conventional serological-based approaches and multiplex PCR for pathogen identification in acute respiratory tract infections.

DNA Microarrays for Pathogen Detection

“…In recent years, a large number of DNA microarray tests for bacterial species identification have been developed for several clinical applications such as sepsis, pneumonia, and infectious diarrhea diagnostics or for the identification of certain pathogens such as Mycobacterium species [10], Escherichia coli [11][12][13][14], chlamydiae [15], and several other bacteria [16][17][18][19][20][21], as shown in Table 4.1. In the following Section 4.2.1 some examples of clinically validated microarray tests will be described in more detail.…”

“…Such arrays may be used for both diagnostic testing and epidemiological investigations. Examples of such arrays for subtyping of influenza A viruses were described by Cannon et al [16] and Heil et al [147]. Several assays were transferred to a microtiterstrip or 96-well plate format allowing simultaneous testing of 8-96 samples [148,149].…”

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

“…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.…”

“…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.…”

An equipment-free polydimethylsiloxane microfluidic spotter for fabrication of microarrays

“…[12][13][14][15] In addition, with the spread of microarray technology, there is increasing demand for the production of different specialized microarrays with a lower density of probes for use in ordinary laboratories. [16][17][18] Although all of the three above technologies are used to produce commercially available microarrays, there are few methods to economically and flexibly produce small batches of custom microarrays for prototyping experiments and specialized applications. To overcome these limitations posed by the conventional technologies used to manufacture microarray, several groups have recently introduced microfluidic printing techniques for fabricating DNA or protein microarrays in a cost-effective and flexible manner, including lineprinting and spot-printing.…”