key: cord-0034682-rb64cvj3 authors: Miszczak, Fabien; Kin, Nathalie; Tesson, Vincent; Vabret, Astrid title: Real-Time RT-PCR Detection of Equine Coronavirus date: 2015-09-10 journal: Animal Coronaviruses DOI: 10.1007/978-1-4939-3414-0_8 sha: c82cbad0be72eb11f029c28f3086d872a84146df doc_id: 34682 cord_uid: rb64cvj3 Equine coronavirus (ECoV) is a recently identified equine virus, involved mainly in enteric infections. Since the ECoV discovery in 1999, only two real-time RT-PCRs have been developed for viral identification. In this chapter we describe a one-step real-time RT-PCR that has been routinely used in our laboratory for ECoV detection from fecal and respiratory samples. Equine coronavirus (ECoV) is a Betacoronavirus-1 in the lineage A (betacoronavirus A1) that was identifi ed at the end of the last century [ 1 ] . ECoV belongs to the genus Betacoronavirus and is closely related to human coronavirus OC43 (HCoV-OC43), bovine coronavirus (BCoV), canine respiratory coronavirus (CRCoV), bubaline coronavirus (BuCoV), and porcine hemagglutinating encephalomyelitis virus (PHEV). ECoV was fi rst isolated in North Carolina, USA, from the feces of a diarrheic foal in 1999 (ECoV-NC99) [ 2 ] . Multiple ECoV outbreaks have recently been reported in Japan [ 3 , 4 ] and in the USA [ 5 ] . Major clinical signs observed were anorexia, fever, lethargy, leukopenia, and diarrhea, and unspecifi c discrete symptoms that do not lend to rapid diagnosis . ECoV was mainly detected in fecal samples from horses and less frequently in respiratory secretions [ 5 -7 ] . A small number of animals with signs of encephalopathic disease have also been observed during these outbreaks [ 8 ] . The current diagnosis of ECoV infection can be performed using virus isolation, electron microscopy, serology [ 9 ] . A reverse transcription loop-mediated isothermal amplifi cation (RT-LAMP), a non-PCR -based nucleic acid amplifi cation assay, has been recently developed for the detection of ECoV in fecal samples [ 10 ] . ECoV was also identifi ed by molecular methods in feces and respiratory samples of foals with and without enteric disease [ 2 , 11 , 12 ] . Real-time RT-PCR assays can enable a prompt identifi cation of ECoV in respiratory and fecal samples of horses who were at the early stage of disease onset [ 5 , 6 ] 10. Loading block (Qiagen ® ). 11. Rotor-Gene Q ® real-time PCR machine (Qiagen ® ) ( see Note 4 ). The protocol described below is routinely used for ECoV clinical diagnosis in fecal and respiratory samples. Two PCR assays are developed in our laboratory targeting partial M and N genes of the ECoV genome [ 6 ] . They were based on short RNA sequences deduced from the ECoV-NC99 strain [ 13 ] . The protocol described is based on the highest sensitive PCR targeting the M gene and proved to be a sensitive and useful tool for ECoV detection in fi eld samples ( see Note 5 ). 1. Transfer 10-50 % of fecal sample into a 2 ml sterile microcentrifuge tube and adjust to 1 ml with autoclaved 1× PBS buffer. 2. Centrifuge at 3,000 rpm (850 × g ) for 30 min at room temperature. 3. Transfer supernatant into a new 1.5 ml sterile microcentrifuge tube. 4. Centrifuge at 10,000 rpm (9,400 × g ) for 15 min at room temperature. 5. Collect supernatant and store at +4 °C until RNA extraction. Respiratory samples (nasopharyngeal swabs) too mucous to be directly extracted with the QIAsymphony ® SP automated instrument are previously treated by proteinase K. 1. Add 10 % of proteinase K to the fi nal volume of respiratory specimen in a sterile 2 ml tube. 2. Briefl y vortex tubes and incubate at 56 °C for 15 min. 3. Store at +4 °C until RNA extraction. 4. Load the required elution rack into the "Eluate" drawer, and load the required reagent cartridge(s) and consumables into the "Reagents and Consumables" drawer. Place the samples into the appropriate sample carrier and the tubes containing the carrier RNA-Buffer AVE mixture into the tube carrier. 6. Enter the required information for each batch of samples to be processed: (a) Sample information. (b) Protocol to be run ("complex200_V6_DSP"). (c) Elution volume (60 μl) and output position. (d) Tubes containing the carrier RNA-Buffer AVE mixture. 7. Run the purifi cation procedure. 8. After the RNA purifi cation, store the purifi ed RNA at 2-8 °C during 24 h before the one-step real-time RT-PCR . For longterm storage of over 24 h, store purifi ed RNA at -20 or -80 °C. 1. Prepare a one-step RT-PCR master mix suffi cient for the designated number of samples in a sterile 1.5 ml microcentrifuge tube on ice, according to Table 2 . Include at least one negative control (autoclaved RNase-free water) and one positive control ( see Note 6 ) for each run. Add additional controls (e.g., purifi ed RNA from the studied samples) as necessary. 2. Insert the strip tubes on the loading block. Aliquot 20 μl of the master mix into separate 0.1 ml strip tubes and label the tubes accordingly. 3. Add 5 μl of each sample and positive control to these tubes. For the negative control, add 5 μl of autoclaved RNase-free water. 4. Close the strip tubes with caps. Insert the strip tubes into the 72-well rotor and lock the rotor into place on the rotor hub of the Rotor-Gene Q ® PCR machine. 5. Turn on the real-time PCR machine (Rotor-Gene Q ® ). Open the "Rotor-Gene Q ® series software". 6. Check the "Locking Ring Attached" checkbox and then click "Next". 7. Set the thermal cycle conditions according to Table 3 . 8. Run the real-time RT-PCR under the conditions shown. 9. In the "Edit Samples" window, input the necessary information for the corresponding samples (e.g., name of the clinical specimen, positive and negative controls). Table 3 Conditions for the one-step real-time RT-PCR assay Step Temperature (°C) Time 10. After performing the RT-PCR , examine the amplifi cation curves of the reactions and the corresponding threshold cycles (Ct). Positive clinical samples will generate amplifi cation curves above the threshold line, and negative samples and water control will be, by contrast, below the threshold line (Fig. 1a ) . Based on the Ct values from tenfold serial dilutions of a reference standard, the RT-PCR can be used to quantify the amount of input target in the positive samples by comparison with the reference (Fig. 1b ) . This amount can be automatically calculated by the software. 3. Primers and probe used in these assays are perfectly matched with the sequences deduced from the original ECoV-NC99 strain (EF446615) [ 13 ] . 4. The RT-PCR has also been validated on the SmartCycler II ® real-time PCR system (Cepheid ® ) with the same RT-PCR thermal cycling conditions. The "M qRT-PCR " should be tested with an internal quality control for viral diagnosis in order to exclude false negatives due to possible inhibition. 6 . A RNA transcript (10 5 copy/μl) deduced from M gene sequence of ECoV-NC99 strain has been used as positive control. Coronavirus diversity, phylogeny and interspecies jumping Characterization of a coronavirus isolated from a diarrheic foal Isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the NC99 strain Epidemic of equine coronavirus at Obihiro Racecourse, Hokkaido, Japan in 2012 Emerging outbreaks associated with equine coronavirus in adult horses First detection of equine coronavirus (ECoV) in Europe Prevalence of equine coronavirus in nasal secretions from horses with fever and upper respiratory tract infection Disease associated with equine coronavirus infection and high case fatality rate Viral diarrhea Rapid detection of equine coronavirus by reverse transcription loop-mediated isothermal amplifi cation Neonatal enterocolitis associated with coronavirus infection in a foal: a case report Infectious agents associated with diarrhoea in neonatal foals in central Kentucky: a comprehensive molecular study Genomic characterization of equine coronavirus We acknowledge French equine practitioners and the LABEO Frank Duncombe Laboratory for providing fecal and respiratory samples needful to develop this real-time RT-PCR .