key: cord-0036109-dmb42kna authors: Vemulapalli, Ramesh title: Real-Time Reverse Transcription Polymerase Chain Reaction for Rapid Detection of Transmissible Gastroenteritis Virus date: 2015-09-10 journal: Animal Coronaviruses DOI: 10.1007/978-1-4939-3414-0_10 sha: 9e8479f3f693499f502384fd441ea492768775c6 doc_id: 36109 cord_uid: dmb42kna Transmissible gastroenteritis (TGE) is a highly contagious disease of pigs caused by the TGE virus (TGEV). Rapid detection of the virus in the affected pigs’ feces is critical for controlling the disease outbreaks. The real-time RT-PCR assay described in this chapter can quickly detect the presence of TGEV in fecal samples with high sensitivity and specificity. Transmissible gastroenteritis (TGE) is a highly contagious, acute viral disease of pigs [ 1 ] . TGE can affect pigs of all ages, but the disease severity and mortality rate are high in piglets under 2 weeks of age. The causative agent is TGE virus (TGEV), a coronavirus that primarily infects and replicates in the epithelial cells of pig intestines. Affected animals shed the virus in their feces . The disease transmission occurs primarily via fecal-oral route [ 2 ] . During an outbreak, rapid detection of TGEV in feces is very useful for implementing the disease management practices in a timely manner. Any TGEV-specifi c diagnostic assay must be able to differentiate it from porcine respiratory coronavirus (PRCV), a natural mutant of TGEV with truncated spike protein and altered cell tropism towards respiratory epithelial cells [ 3 ] . PRCV mostly causes mild or subclinical respiratory disease. However, in some PRCV-infected pigs, the virus can be shed in the feces [ 4 ] . Realtime PCR -based assays are well suited for rapid, specifi c, and sensitive detection of viruses such as TGEV. The real-time RT-PCR assay described here is based on amplifi cation of a conserved region of the spike protein gene of TGEV strains and detection of the amplifi ed products using a TaqMan probe [ 5 ] . The assay, along with the RNA extraction method described here, can be established in any molecular diagnostic laboratory for detection of TGEV in pig fecal samples [ 6 -8 ] . Preventing contamination of samples and reagents with nucleic acids and nucleases is critical to obtaining accurate and reproducible results with any PCR -based diagnostic assay. It is recommended that the nucleic acid extraction, preparation of master mix, and real-time PCR amplifi cation are performed in three physically separated areas. Each of these areas should have separate set of laboratory instruments and supplies that are to be used only in their assigned location [ 9 ] . Including a positive extraction control and a negative extraction control along with each batch of clinical samples is recommended to monitor the effi ciency of RNA extraction and potential cross contamination of the samples [ 9 ] . PCR inhibitors are often co-extracted with nucleic acids from fecal samples. In our experience, RNA extracted from pig fecal samples using the following method is suitable for TGEV detection using the real-time RT-PCR assay. Other extraction methods that produce PCR inhibitor-free RNA can also be used ( see Note 1 ). The presence of PCR inhibitors in the extracted can be monitored by using an internal control ( see Note 2 ). 1. Prepare a 20 % (w/v) suspension of feces in DNase/RNasefree distilled water. Feces in liquid form can be used directly. 2. Transfer 250 μl of the fecal suspension into a 1.5 ml microcentrifuge tube. 3. Add 750 μl of TRIzol LS reagent, briefl y vortex the tube for 10 s, and incubate at room temperature for 5 min. 4. Add 200 μl of chloroform to the tube. Vortex the tube for 5 s and incubate at room temperature for 3 min. 5. Centrifuge the tube at 12,000 × g for 10 min. 11. Repeat step 10 for a second wash of the spin column with RPE buffer. 12. Place the spin column into a new collection tube and centrifuge at 10,000 × g for 1 min to dry the membrane of the column. 13. Place the spin column in a 1.5 ml microcentrifuge tube and add 30 μl of DNase/RNase-free water to the column. Incubate at room temperature for 1 min. 14. Centrifuge at 10,000 × g for 1 min. 15. Discard the spin column and store the eluted RNA at 4 °C if it is used in the real-time RT-PCR assay within 12 h or at −20 °C if it is used after 12 h ( see Note 4 ). 1. Turn on the real-time PCR machine. Follow the software directions of the machine manufacturer to confi rm that the FAM signal data will be gathered during the amplifi cation. Program the thermal cycle and data collection conditions according to Table 1 . 2. Prepare a master mix suffi cient for the intended number of samples in a sterile 1.5 ml microcentrifuge tube according to Table 2 . Add at least a no-template control (NTC) and a positive amplifi cation control (PAC) to the number required reactions. 3. Close the cap of the microcentrifuge tube. Vortex and centrifuge the tube briefl y. 4. Aliquot 20 μl of the master mix into each PCR reaction tube. Table 1 Thermal cycling conditions Step Temperature (°C) Time DNase/RNase-free distilled water Feces samples from suspected pigs RNasin Ribonuclease Inhibitor (Promega) or equivalent mM MgCl 2 solution for PCR (Sigma-Aldrich) or equivalent DNase/RNase-free distilled water μM Forward primer CCA(BHQ1)-3′(FAM, 6-carboxyfl uorescein; BHQ1, black hole quencher 1; Eurofi ns MWG Operon) Real-time PCR machine, such as Smart Cycler II (Cepheid), 7300 Real-Time PCR System (Applied Biosystems), or equivalent PCR reaction tubes suitable for the real-time PCR machine platform Transmissible gastroenteritis Immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine Porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions Respiratory and fecal shedding of porcine respiratory coronavirus (PRCV) in sentinel weaned pigs and sequence of the partial S-gene of the PRCV isolates A real-time TaqMan ® RT-PCR assay with an internal amplifi cation control for rapid detection of transmissible gastroenteritis virus in swine fecal samples Attempted experimental reproduction of porcine periweaning-failure-to-thrive syndrome using tissue homogenates The fi rst case of porcine epidemic diarrhea in Canada A novel watery diarrhea caused by the co-infection of neonatal piglets with Clostridium perfringens type A and Escherichia coli (K88, 987P) Quality assurance and quality control in the routine molecular diagnostic laboratory for infectious diseases