In brief, 90 μl of viral RNA was eluted from rectal swabs, fecal samples and oral fluid specimens using the Ambion® MagMAX™ viral RNA isolation kit (Life Technologies, Carlsbad CA USA) and a KingFisher® 96 magnetic particle processor (Thermo-Fisher Scientific) following the procedures provided by the manufacturers. Samples were tested for PEDV using a PEDV N gene-based rRT-PCR described in Madson et al. [7 (link)] and performed routinely at the Iowa State University-Veterinary Diagnostic Laboratory (ISU-VDL SOP 9.5263). The forward primer sequence was 5′-CGCAAAGACTGAACCCACTAACCT-3′, the reverse primer sequence was 5′-TTGCCTCTGTTGTTACTTGGAGAT-3′, and probe sequence was 5′-FAM-TGTTGCCAT/ZEN/TACCACGACTCCTGC-Iowa Black-3′. The eluted RNA, primers, and probe were mixed with commercial reagents TaqMan® Fast Virus 1-Step Master Mix (Life Technologies) and the rRT-PCR reactions were conducted on an ABI 7500 Fast instrument (Life Technologies) in fast mode as follows: 1 cycle at 50 °C for 5 min, 1 cycle at 95 °C for 20 s, 40 cycles at 95 °C for 3 s, and 60 °C for 30 s. The results were analyzed using an automatic baseline setting with a threshold at 0.1. Quantification cycle (Cq) values < 35 were considered positive for the corresponding coronavirus. Data were reported as ‘adjusted Cqs’:
Abi 7500 fast instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom
The ABI 7500 Fast instrument is a real-time PCR system designed for fast and accurate nucleic acid quantification. It features a Peltier-based thermal cycler and a high-performance optical detection system to enable efficient and precise real-time PCR analysis.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Rt2 first strand kit, by Qiagen (7 mentions)
Reverse transcriptase premix, by Elpis Biotech (6 mentions)
Nucleospin rna 2 kit, by Macherey-Nagel (5 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Path id multiplex one step rt pcr kit, by Thermo Fisher Scientific (3 mentions)
Power sybr green polymerase chain reaction master mix, by Thermo Fisher Scientific (3 mentions)
M mlv transcriptase, by Takara Bio (3 mentions)
Magmax viral pathogen extraction kit, by Thermo Fisher Scientific (3 mentions)
Kingfisher flex machine, by Thermo Fisher Scientific (3 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (3 mentions)
Paxgene blood rna tube, by Qiagen (3 mentions)
Rt2 profiler pcr array human oxidative stress plus, by Qiagen (3 mentions)
Power sybr green pcr master mix reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
7500 software v2, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Superscript reverse transcriptase, by Takara Bio (2 mentions)
Human crohn s disease rt2 profiler pcr array, by Qiagen (2 mentions)
66 protocols using abi 7500 fast instrument
1
PEDV RNA Extraction and Detection
2
Quantitative mRNA Assessment by RT-PCR
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Quantitative assessment of mRNA levels was performed by real-time RT-PCR on an ABI 7500 Fast instrument with Power SYBR Green PCR Master Mix reagent (Life Technologies). Real-time conditions were as follows: 95°C (15 sec), 40 cycles at 95°C (15 sec), and 60°C (1 min). According to melting point analysis, only one PCR product was amplified under these conditions. The relative quantity of a target, normalized to the endogenous control β-2 microglobulin and β-actin as internal calibrators, was calculated as the fold difference and further processed using statistical analysis.
3
Real-Time RT-PCR Detection of Coronaviruses
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The eluted RNA, primers, and probe were mixed with commercial reagents (Path-ID® Multiplex One-Step RT-PCR kit, Life Technologies) and the RT-PCR reactions were conducted on an ABI 7500 Fast instrument (Life Technologies) as follows: 48°C for 10 min, 95°C for 10 min, 95°C for 15 s (45 cycles) and 60°C for 45 s. The real-time RT-PCR (rRT-PCR) results were analyzed using an automatic baseline setting with a threshold at 0.1. Quantification cycle (Cq) values ≤ 35 were considered positive for the corresponding coronavirus. Data were reported as "adjusted Cq":
4
Multiplex Real-Time PCR for Respiratory Pathogens
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The multiplex real-time PCR FTD assay was performed on an ABI 7500 Fast instrument (Life Technologies, USA) as per the manufacturer’s instructions using an AgPath-ID ™ One-Step RT-PCR kit (Ambion) with the FTD Respiratory pathogens 21 kit (Fast Track Diagnosis, Luxembourg) for the detection of 18 viruses using five tubes containing primer and probe mix for different viruses; Tube-1 [Influenza A (Flu A), Influenza A subtype H1N1 (Pandemic H1N1), human Rhinovirus (HRV), Influenza B (Flu B)], Tube-2 [human Coronaviruses NL63 (HCoV-NL63), 229E (HCoV 229E), OC43 (HCoV-OC43), and HKU1 (HCoV HKU1)], Tube-3 [human Parainfluenza viruses, 2, 3, and 4 (HPIV- 2, 3 and 4) & IC], Tube-4 [human Parainfluenza viruses-1, Mycoplasma pneumoniae (M.pneu), human Bocavirus (HBoV), human Metapneumovirus (HMPV A/B)] and Tube-5 [Respiratory Syncytial virus (RSVA/B), human Adenovirus (HAdV), Enterovirus (EV), human Parechovirus (HPeV)]. The multiplex real time RT-PCR thermal profile for the FTD kit was as follows; 50 °C for 15 min, 95 °C for 10 min, 40 cycles of 95 °C for 8 s, 60 °C for 34 s, whereas the thermal profile for the custom assay was set at 50 °C for 30 min, 95 °C for 10 min, 45 cycles of 95 °C for 15 s, 55 °C for 30 s.
5
Real-time RT-PCR for BVDV Genotyping
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Genotyping was conducted using a commercial real time RT-PCR kit (Cador BVDV Type 1/2 real time RT-PCR Kit, Qiagen, UK) capable of distinguishing between BVDV-1, BVDV-2 and BDV in a triplex reaction. The assay was run on an ABI 7500 Fast instrument (Life Technologies) according to the manufacturer’s instructions except that the reaction volume comprised 20 μl of mastermix and 5 μl RNA.
6
Multiplex Real-Time PCR for Respiratory Pathogens
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The multiplex real-time PCR FTD assay was performed on an ABI 7500 Fast instrument (Life Technologies, Carlsbad, CA, USA) as per the manufacturer’s instructions using an AgPath-ID™ One-Step RT-PCR kit (Ambion Inc., Austin, TX, USA) with the FTD Respiratory pathogens kit (Fast Track Diagnosis) for the detection of 12 viruses using five tubes containing primer and probe mix for different viruses; tube-1 [influenza A (flu A), influenza A subtype H1N1 (pandemic H1N1), human rhinovirus (HRV), influenza B (flu B)], tube-2 [human coronaviruses NL63 (HCoV-NL63), 229E (HCoV 229E), OC43 (HCoV-OC43), and HKU1 (HCoV HKU1)], tube-3 [human parainfluenza viruses, 2, 3, and 4 (HPIV- 2, 3 and 4) & IC], tube-4 [human parainfluenza viruses-1, human bocavirus (HBoV), human metapneumovirus (HMPV A/B)] and tube-5 [respiratory syncytial virus (RSVA/B), human adenovirus (HAdV). The multiplex real-time RT-PCR thermal profile for the FTD kit was as follows; 50°C for 15 min, 95°C for 10 min, 40 cycles of 95°C for 8 s, 60°C for 34 s, whereas the thermal profile for the custom assay was set at 50°C for 30 min, 95°C for 10 min, 45 cycles of 95°C for 15 s, 55°C for 30 s.
7
Quantitative PDCoV Detection in Samples
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Rectal swabs, serum and various tissues were tested by a PDCoV M-gene based real-time RT-PCR including viral standards with known infectivity titers for quantification. Briefly, viral RNA was extracted from rectal swabs, serum, and 10% tissue homogenates as previously described (Chen et al., 2014 (link)). Five µl of each template was used in PCR setup in a 25 µl total reaction using Path-ID™ Multiplex One-Step RT-PCR Kit (Life Technologies, Carlsbad, CA) and primers (forward primer 5′-CGACCACATGGCTCCAATTC-3′, reverse primer 5′-CAGCTCTTGCCCATGTAGCTT-3′) and probe (5′-CACACCAGTCGTTAAGCATGGCAAG C-3′). The probe was labeled using the FAM/ZEN/3′Iowa Black detector (Integrated DNA Technologies, Coralville, IA). The RT-PCR was run on an ABI 7500 Fast instrument (Life technologies, Carlsbad, CA) with the following conditions: 1 cycle of 48 °C for 10 min, 1 cycle of 95 °C for 10 min, and 45 cycles of 95 °C for 15 s and 60 °C for 45 s. A PDCoV isolate with known infectivity titer was 10-fold serially diluted for generating a standard curve in each PCR plate. Virus concentration (TCID50/ml) in tested samples was calculated based on the standard curve. The mean cycle threshold (Ct) values were calculated based on PCR positive samples, and the mean virus titers were calculated based on all pigs within the group.
8
Duplex Real-Time RT-PCR for Norovirus Detection
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Real-time RT-PCR was performed in duplexed reactions by combining primers and probes commonly used for GI ad GII noroviruses (Additional file 2 : Table S1). Briefly, PCR amplifications were performed on a Life Technologies ABI 7500 Fast instrument in 25 μl reactions consisting of: SuperScript III Platinum One-Step 1× master mix (Life Technologies), 0.2 μl enzyme mix, 20U RNaseOUT, and 400 nM of each primer and probe (Additional file 2 : Table S1). Amplification conditions were as follows: 50°C for 30 min; 95°C for 30s; and 45 cycles of 95°C for 30s and 60°C for 1 min. Ct values were determined using the manufacturer's software (version 2.0.5).
9
Gut Microbiome Quantification in Mice
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Genomic DNA was extracted from mouse fecal samples using QIAamp Fast DNA Stool Mini kits (Qiagen, Germantown, MD, USA), quantified using a Take3 plate and Synergy H1 microplate spectrophotometer (BioTek, Sunnyvale, CA, USA), and adjusted to a final concentration of 1 ng/μL. Quantitative real-time PCR was performed on an ABI 7500 Fast instrument (Life Technologies, Carlsbad, CA, USA) in a total volume of 20 μL containing 10 μL 2× SYBR Green PCR Master Mix, 1 μL of each primer from the GUt Low-Density Array (GULDA) [25 (link)], 4.4 μL of nuclease-free water, and 3.6 μL of template DNA. The amplification program consisted of 50 °C for 2 min; 95 °C for 10 min; and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. A dissociation curve was recorded at 95 °C for 15 s and 60 °C for 15 s; then, the temperature was increased to 95 °C at a 2% rate). The mean Ct value was determined based on a set threshold value of 0.2 and using automatic baseline correction. Differences in Ct values for each bacterial target (N0 normalization) were calculated between those obtained with the universal and target-specific primers and log-transformed. Fold changes for target amplicons were calculated as the (log 2) ratio of normalized abundances and determined as a percentage of the microbiome composition.
10
Duplex real-time RT-PCR for Norovirus
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Real-time RT-PCR was performed in a duplex reaction by combining primers and probes commonly used for GI ad GII noroviruses [3 (link)]. Briefly, PCR amplifications were performed on a Life Technologies ABI 7500 Fast instrument in 25 μL reactions consisting of the following: SuperScript III Platinum One-Step 1x master mix (Life Technologies), 0.2 μL enzyme mix, 20 U RNaseOUT, and 400 nM of each primer and probe (Table S1). Amplification conditions were as follows: 50°C for 30 min; 95°C for 30 s; and 45 cycles of 95°C for 30 s and 60°C for 1 min. Ct values were determined using the manufacturer's software (version 2.0.5).
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