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Syracuse University

- Bacterial culture

- Viral detection (using PCR)

- Cytokine measurement (using quantitative PCR)

- RNA-sequencing 

- 16S ribosomal RNA gene sequencing

Multiple Tests that can be Performed with NP Swab

Lopez, S.M.C., Martin, J.M., Johnson, M. et al. A method of processing nasopharyngeal swabs to enable multiple testing. Pediatr Res 86, 651–654 (2019). https://doi-org.libezproxy2.syr.edu/10.1038/s41390-019-0498-1
https://www.nature.com/articles/s41390-019-0498-1

A method of processing nasopharyngeal swabs to enable multiple testing

Cultures were incubated overnight at 37 °C with 5% CO2, and if no growth was present in a culture after this time, it was reincubated for another 24 hours. Researchers used standard techniques (assessment of growth in 0, 1, 2, 3, or 4 quadrants) to measure the presence of pathogenic bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Growth in 3+ and 4+ quadrants was identified as heavy growth.


Method for Testing Bacterial Culture with NP Swab

"Nucleic acid extraction was performed with the ABI MagMax96 Express automated instrument and the MagMax Viral Isolation Kit. Individual real-time reverse transcriptase (RT-PCR) assays for the detection and subtyping of adenovirus, influenza subtypes A/B/C, human metapneumovirus, human rhinovirus, parainfluenza virus subtypes 1–4, and respiratory syncytial virus were performed. All specimens were tested for RNase P to confirm RNA integrity and monitor for PCR inhibitors. Samples were run in batches on an ABI Step One Plus 96-well real-time PCR instrument. Negative and positive controls were included with each run, including RNA runoff transcripts to generate a quantitation curve. Specimens were considered positive if the Ct value was <40 cycles."


Method for Viral Detection with NP Swab

"Nucleic acid extraction was performed with the ABI MagMax96 Express automated instrument from one of the four aliquots prepared from the original sample. Gene expression of interleukin-6 (IL-6), IL-10, IL-13, IL-17, IL-25, and IL-33; interferon: α, β, γ, and λ; and IL-8 was measured using exon-spanning primers and probes according to the manufacturer’s instructions (TaqMan™, Applied Biosystems). All values were normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) housekeeping gene."

Method for Cytokine Measurement with NP Swab

"RNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s protocol from sample prepared from the tip of the swab. RNA purity was assessed using Qubit 2.0 fluorometer and Agilent TapeStation 2200 for RNA quantity and quality. Host RNA-seq libraries were generated using Illumina TruSeq RNA Access, which converts total RNA into template molecules, followed by sequence-specific capture of coding RNA. The complementary DNA (cDNA) libraries were quantified using Qubit 2.0 fluorometer, and the quality was examined using Agilent Tapestation 2200.The cDNA libraries were pooled at a final concentration of 2.0 pM. Cluster generation and paired-read, dual-indexed 75 bp sequencing was performed on Illumina NextSeq 500."

Method for RNA Sequencing with NP Swab

"Total nucleic acids were extracted from a frozen specimen without a buffer using the DNeasy UltraClean DNA Extraction Kit (Qiagen) and quantified using the Qubit™ assay (Invitrogen) from one of the four aliquots prepared from the original sample. The 16S rRNA gene V4–V5 region was amplified using a modified 515F–806R primer pair.20 Sequencing of amplicons was completed using the Illumina MiSeq platform to generate 2 × 250 paired-end reads."

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