Total nucleic acid (TNA) was extracted from 400 µL of Universal Transport Media containing the NP and OP specimens and eluted into 110 µL using the EZ1 Advanced XL Instrument with the EZ1 Mini Viral 2.0 Kit (Qiagen) following the manufacturer's instructions. Seven specimens were extracted at a time with an extraction control consisting of nuclease‐free water.

The TNA extracts from NP/OP specimens were screened individually for detection of respiratory pathogens using eight multiplex reverse‐transcriptase real‐time polymerase chain reactions (rRT‐PCR) from the FTD‐33 Test Kit (Fast Track Diagnostics). The kit is used for detection of the following respiratory viruses, bacteria, and fungi: influenza A, influenza A subtype A(H1N1)pdm09, influenza B, and influenza C; parainfluenza viruses 1, 2, 3, and 4; coronaviruses NL63, 229E, OC43, and HKU1; human metapneumoviruses A and B; rhinovirus; respiratory syncytial viruses A and B; adenovirus; enterovirus; parechovirus; bocavirus; Pneumocystis jirovecii; Mycoplasma pneumoniae; Chlamydia pneumoniae; Streptococcus pneumoniae; Haemophilus influenzae; Haemophilus influenzae type B; Staphylococcus aureus; Moraxella catarrhalis; Bordetella species (excluding Bordetella parapertussis); Klebsiella pneumoniae; Legionella species; and Salmonella species. 6

The eight multiplex rRT‐PCR reactions were set up following the manufacturer's instructions. 6 Each reaction mix consisted of 10 µL TNA, 1.5 µL of oligonucleotide mix, 12.5 µL 2 X AgPath‐IDTM One‐Step RT‐PCR buffer and 1 µL 25 X AgPath‐IDTM One‐Step RT‐PCR Enzyme Mix (Thermo Fisher Scientific). The manufacturer's instructions recommend introducing exogenous internal control material provided with the FTD‐33 Kit into each clinical specimen prior to nucleic acid extraction and testing with corresponding rRT‐PCR oligonucleotide mix to monitor for PCR inhibition. This step was excluded to minimize potential contamination of primary specimen material. Instead, each specimen was tested in parallel for the presence of human ribonuclease P (RNase P). Detection of RNase P, which is ubiquitous in human cells, serves as a control for specimen extraction and PCR inhibition without manipulation of primary specimens. Reaction mixtures containing a no‐template control, extraction control, and positive control (Resp21 PC or Resp33 PC2) were included for each multiplex reaction mix. An extraction control and no‐template control materials were included for testing of the eight multiplex reaction mixes and the RNase P reaction mix. The positive control (Resp21 PC or Resp33 PC2) consisting of pooled plasmids 6 from the FTD‐33 Kit was also included for testing of the multiplex reaction mixes and human DNA was included as the positive control for the RNase P reaction mix. All assays were tested using the Applied Biosystems 7500 Real‐Time PCR Instrument (Thermo Fisher Scientific) with the following cycling conditions: 42°C for 15 minutes, 94°C for 3 minutes, 40 cycles of 94°C for 8 seconds, and 60°C for 34 seconds. Any assay or specimen with a control result deviating from the expected result was retested.

Influenza viruses were detected using singleplex reverse‐transcriptase real‐time polymerase chain reaction (rRT‐PCR) methods from the Centers for Disease Control and Prevention (CDC) Influenza Division (Atlanta, GA, USA) for typing and subtyping of influenza A and influenza B. 4 Samples were first screened for influenza A and B viruses. The typing kit includes influenza A, influenza B, and human RNase P primers and probes. Specimens found to be positive for influenza A virus were subtyped using an influenza A(H3/H1pdm09) panel. For influenza B virus–positive samples, an influenza B lineage genotyping panel including B/Victoria and B/Yamagata primers and probes was used. Each reaction mix consisted of 5 µL total nucleic acid, 5 of nuclease‐free water, 0.5 µL of each primer (forward and reverse) and probe, 12.5 µL 2 X AgPath‐IDTM One‐Step RT‐PCR buffer, and 1 µL 25 X AgPath‐IDTM One‐Step RT‐PCR enzyme mix (Thermo Fisher Scientific). Reaction mixtures containing a no‐template control (negative control), extraction control, human specimen control, and pooled influenza‐positive control were included for each singleplex reaction mix. The rRT‐PCR testing was performed at a final volume of 25 µL on the Applied Biosystems 7500 Real‐Time PCR Instrument with the following cycling conditions: 50°C for 30 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 55°C for 30 seconds. Any assay or specimen with a control result deviating from the expected result was retested.

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