A novel real-time PCR assay panel for detection of common respiratory pathogens in a convenient, strip-tube array format

Abstract

A B S T R A C TCommercial multiplex assays, built on different chemistries and platforms are widely available for simultaneous detection of pathogens that cause respiratory infections. However, these tests are often difficult to implement in a resource limited setting because of high cost. In this study, we developed and validated a method for simultaneous testing of common respiratory pathogens (Respanel) by real-time PCR in a convenient, strip-tube array format. Primers and probes for sixteen PCR assays were selected from the literature or newly designed. Following optimization of individual PCR assays, strip-tube arrays were prepared by dispensing primer-probe mixes (PPM) into two sets of 8-tube strips. Nucleic acid extracts from specimens were mixed with PCR master mix, and dispensed column-wise into 2 × 8-wells of a 96-well plate. PPMs from strip-tubes were then added to the wells using a multichannel pipette for real-time PCR. Individual PCR assays were optimized using previously known specimens (n = 394) with 91%-100% concordance with culture, DFA or PCR results. Respanel was then tested in a routine manner at two different sites using specimens (n = 147) previously tested by Qiagen Resplex I &II or Fast-Track Diagnostics Respiratory Pathogens 21 assays. The sensitivity, specificity and accuracy of Respanel were 94%, 95% and 95%, respectively, against Resplex and 88%, 100% and 99%, respectively, against FTDRP21. Respanel detected more pathogens (p < 0.05) than Resplex but the rate of pathogen detection was not significantly different from FTDRP21. Respanel is a convenient and inexpensive assay that is more sensitive than Resplex and comparable to FTDRP21 for the detection of common respiratory pathogens.

Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA

“…RT-qPCR: Primers and probes for detection of SARS-CoV-2, HCoV-HKU1, RNaseP and MS2 bacteriophage are listed in S1 Table [4,[12][13][14]. For detection of HCoV-HKU1 using Quantifast Pathogen RT-PCR + IC kit (Qiagen), five μl of extracted or pre-treated samples were mixed with 20 μl of a master mix containing 5 μl of Quantifast Pathogen RT-PCR + IC Master Mix, 0.25 μl of Quantifast RT Mix and 0.5 μl of 50 x ROX dye solution and primers and probes to final concentrations shown in S1 Table. Thermal cycling was performed in a ABI7500 Fast instrument (Thermofisher Scientific) with 1 cycle of reverse transcription at 50˚C for 20 min followed by 1 cycle of PCR activation at 95˚C for 5 min, followed by 40 amplification cycles each consisting of 95˚C-15s and 60˚C-60s.…”

Correlation between the Antibiotic Resistance Genes and Susceptibility to Antibiotics among the Carbapenem-Resistant Gram-Negative Pathogens

“…This technology identifies the nucleic acids in the samples even if the pathogen is fastidious or the patient received prior antimicrobial therapy which would render the culture results incomprehensive [ 57 ]. Other rapid molecular diagnosis techniques include the RespiFinder ® SMART 22 FAST, the Unyvero pneumonia cartridge, the ResPlex TM Panels, scalable target analysis routine (STAR) technology and PLEX-ID technology [ 58 , 59 , 60 , 61 ]. Unfortunately, these techniques are not widespread in Egyptian hospitals as they are much more expensive.…”

Design and Evaluation of a Macroarray for Detection, Identification, and Typing of Viral Hemorrhagic Septicemia Virus (VHSV)

“…Venter et al [21] designed a macroarray for diagnosis of meningoencephalitis, mostly of viral etiology; however, that procedure actually requires an initial application of PCR to the extracted nucleic acid, and then the array is used just for the detection by NAH. The real concept of a PCR-based macroarray has been applied by Hasan et al [20] for respiratory pathogens, and by Ries et al [19] for bluetongue virus. In both cases, 96-well PCR plates are filled with the corresponding mixture of primers and probes, in a solution, and the plates can be stored at −20 • C until use; however, prior to their use, the plates must be thawed and briefly centrifuged.…”

“…However, since it is based on nucleic acid hybridization (NAH), and because the handicap of NAH is its relative low sensitivity [18], it would not be an appropriate option for diagnosis of this virus, especially in asymptomatic fish. Nevertheless, its combination with PCR amplification solves this drawback, and thus it has been widely applied in recent studies using both macro and microarrays for the diagnosis of viral diseases [19][20][21][22][23][24].…”