key: cord-016179-4i1n9j4x
authors: Chen, Yi-Ning; Wu, Ching Ching; Lin, Tsang Long
title: Real-Time Reverse Transcription-Polymerase Chain Reaction for Detection and Quantitation of Turkey Coronavirus RNA in Feces and Intestine Tissues
date: 2015-09-10
journal: Animal Coronaviruses
DOI: 10.1007/978-1-4939-3414-0_13
sha: 
doc_id: 16179
cord_uid: 4i1n9j4x

Turkey coronavirus (TCoV) infection causes acute atrophic enteritis in turkey poults, leading to significant economic loss in the turkey industry. Rapid detection, differentiation, and quantitation of TCoV are critical to the diagnosis and control of the disease. A specific one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using TCoV-specific primers and dual-labeled fluorescent probe for detection and quantitation of TCoV in feces and intestine tissues is described in this chapter. The fluorogenic probe labeled with a reporter dye (FAM, 6-carboxytetramethylrhodamine) and a quencher dye (Absolute Quencher™) was designed to bind to a 186 base-pair fragment flanked by the two PCR primers targeting the 3′ end of spike gene (S2) of TCoV. The assay is highly specific and sensitive and can quantitate between 10(2) and 10(10) copies/mL of viral genome. It is useful in monitoring the progression of TCoV-induced atrophic enteritis in the turkey flocks.

Turkey coronavirus (TCoV) causes atrophic enteritis in turkeys and outbreaks or cases of turkey coronaviral enteritis occurred and still occurs in the USA [1] , Canada [2] , Brazil [3] , and Europe [4] . TCoV belongs to species Avian coronavirus of the genus Gammacoronavirus in the family Coronaviridae. The genome of TCoV is a linear positive-sense single-stranded RNA encoding three major structural proteins including spike (S), membrane (M), and nucleocapsid (N) protein. The amino terminal region of S protein (S1) containing receptor-binding domain and neutralizing epitopes can determine host specificity and induce the production of neutralizing antibodies [5] . The carboxyl terminal region of S protein (S2) consisting of transmembrane domain is responsible for cell fusion and virus assembly [6] . S gene is a more common target used for coronavirus (CoV) differentiation because S gene is highly variable among different CoVs while M and N genes are more conserved. Within the S gene, the S2 gene is more conserved than S1 gene between different CoVs and between different isolates or strains of the same CoV [7] . Therefore, S2 gene is chosen as a target to detect TCoV and differentiate TCoV from other CoVs.

There is no cell culture system for TCoV; thus virus isolation is not feasible. Because the sequence information of TCoV is available, reverse transcription-polymerase chain reaction (RT-PCR)-based methods with high specificity and sensitivity have been developed [7, 8] . Real-time RT-PCR illustrated here uses a pair of TCoV-specific primers targeting a 186 base-pair fragment of TCoV S2 gene and a dual-labeled probe with a reporter dye (FAM) and a quencher dye (Absolute Quencher™) combined with the 5′ to 3′ exonuclease activity of Taq polymerase to increase the release of reporter dye fluorescence in the course of PCR amplification [9] . Quantitative data can be accessed by the standard curve established with serial dilutions of standard RNA. The procedure does not need post-PCR electrophoresis, so the processing time can be significantly reduced and the risks for carryover and cross-contamination between samples can be lessened. In this chapter, the protocol for one-step real-time RT-PCR to detect, differentiate, and quantitative TCoV RNA in the feces and intestinal tissue is presented. In step 1, feces or intestine tissues were collected into RNAlater RNA stabilization reagent. In step 2, TCoV RNA was extracted from feces using QIAamp viral RNA mini kit or intestine tissues using RNAeasy mini kit. In step 3, the extracted RNA was subjected to one-step real-time RT-PCR for detection and quantitation of TCoV in feces or intestine tissues. In step 4, a standard curve was established by serially diluted in vitro-transcribed RNA for absolute quantitation of TCoV. (c) Add 30 mL of 96-100 % ethanol into 13 mL of concentrated Buffer AW2.

Real-Time RT-PCR for Turkey Coronavirus RNA 4. RNeasy mini kit (Qiagen, Valencia, CA, USA): The kit contains Buffer RLT (lysis), RW1 (wash), and RPE (elute). Buffer RLT and AW1 contain guanidine thiocyanate, which is harmful. RNA from intestine tissues can bind to silica membrane of RNeasy spin columns with a binding capacity of 100 μg.

(a) Add 10 μL β-mercaptoethanol (β-ME) per 1 mL Buffer RLT in a fume hood. Buffer BLT with β-ME can be stored at room temperature for up to 1 month.

(b) Add 44 mL of 96-100 % ethanol to 11 mL of concentrated Buffer RPE. 1. Use sterile forceps and scissors to cut 0.5 cm long segment of duodenum, jejunum, ileum, or cecum. 8. Add 500 μL of Buffer AW1 and centrifuge at 8000 rpm for 1 min. Place the QIAamp spin column into a clean 2 mL collection tube and discard the tube containing the filtrate. 9 . Add 500 μL of Buffer AW2 and centrifuge at 14,000 rpm (18407 × g) for 3 min. Change the direction of tubes and centrifuge at 14,000 rpm for another 1 min. 10 . Place the QIAamp spin column in a clean 1.7 mL microcentrifuge tube and add 60 μL of Buffer AVE. Incubate at room temperature for 1 min and centrifuge at 8000 rpm for 1 min.

11. Take 4 μL RNA into 96 μL Buffer AVE and measure the amount of RNA by GeneQuant following the manufacturer's instruction.

12. Aliquot the RNA into 5 μL each tube and store in −80 °C freezer for use in real-time RT-PCR.

1. Take intestine segment out of 1.7 mL microcentrifuge tubes containing 1 mL of RNAlater RNA stabilization reagent and put the tissue specimen on top of the plastic paper treated with RNase Away and DEPC H 2 O. 14. Aliquot the RNA into 5 μL tube individually and store in −80 °C freezer for use in real-time RT-PCR.

1. Prepare a total of 25 μL of reaction mixture ( Table 2 ) on ice by using TCoV-specific primers, probe, reaction buffer from Platinum ® Quantitative RT-PCR ThermoScript™ One-Step System, and 5 μL of RNA template. Mix gently by pipetting up and down (see Note 4). 1. Amplify partial S2 gene of TCoV in the region encompassing the fragment targeted by real-time RT-PCR by PCR with forward primer 6F and reverse primer 6R (Table 1 ) and the amplified fragment is designed as 6F/6R fragment. The fragments underlined in primers 6F and 6R are recognized by restriction enzymes NcoI and KpnI, respectively.

2. Double-digest the pTriEx-3 DNA-Novagen (1-3 μg total) or 6F/6R fragment (1-3 μg total) with 1 μL of 1/10 diluted NcoI and 1 μL of 1/10 diluted KpnI (NEB) in 50 μL reaction containing 5 μL of 10× NEB ® Buffer 1 and 5 μL of bovine serum albumin (BSA, 1 mg/mL). Incubate the reaction at 37 °C for 1 h.

3. Ligate the treated pTriEx-3 and 6 F/6R fragment (638 bp) using T4 DNA ligase in 20 μL reaction to become pTriEx3-6F/6R. The reaction solution contains total 64 ng of the 10 6 023 10 9 2 3

1. RNAlater stabilization reagent is not designed for fecal samples but the previous study [10] showed a good stability of viral RNA in turkey feces by using RNAlater reagent. It is very critical to mix feces or cloacal swab with RNAlater to make feces submerged and contact RNAlater reagent completely for the stability of RNA in feces.

2. The thickness of tissue must be less than 0.5 cm. RNA in harvested intestine tissue is not protected until the tissue is completely submerged in a sufficient volume of RNAlater RNA Stabilization Reagent at about 10 μL reagent per 1 mg tissue. The intestinal content may interfere with the reaction of RNAlater reagent, so it is critical to wash off the intestinal contents. Insufficient RNA reagent may cause RNA degradation during storage. It is not recommended to harvest tissues frozen in liquid nitrogen or dry ice and later thaw them for RNAlater storage or RNA extraction because the process would cause severe RNA degradation.

3. The heat produced by the process of hominization can damage the integrity of RNA in intestine tissues leading to low sensitivity of real-time RT-PCR. Therefore, it is recommended to homogenize the intestine tissue for 30 s and cool the tube in ice in cycles until the intestine tissue is homogenized thoroughly.

4. Real-time RT-PCR is a very sensitive assay, so it is very easy to be interfered by bubbles created by pipetting. To use pipettes and take small volume of reagents (<10 μL) precisely, it is recommended to practice before performing the real test. In addition, the nonspecific reactions can be minimized by mixing reagents on the precooled (store at −20 °C freezer) aluminum loading block (Qiagen) on ice.

5. Example of running a set of samples from the setup to the end of calculating the copy number is illustrated below. First, collect ileum and cecum samples in RNAlater solution from three turkeys at 5 days post-infection of TCoV and the same kind of samples from negative control turkeys without TCoV infection. Second, label the samples from turkeys infected with TCoV as d5-i1 to d5-i3 and d5-c1to d5-c3, and those from negative control turkeys without TCoV infection as N-i1 to N-i3 and N-c1 to N-c3. Third, extract RNA from the samples according to Sect. 3.2.2. Next, arrange the samples in 96-well plate for one-step real-time RT-PCR (Table 3) and set up the real-time RT-PCR conditions. Prepare nuclease-free water as template for the non-template control 

Samples are arranged in Row A to H from Column 1 to 12 in one 96-well plate. STD1 to STD8 are tenfold serially diluted standard RNA of pTriEx3-6F/6R from 10 2 to 10 9 copies/μL. NTC is nuclease-free water as template for realtime RT-PCR (NTC) and tenfold serially diluted standard RNA of pTriEx3-6F/6R from 10 2 to 10 9 copies/μL, as STD1 to STD8. After the reaction complete, establish the standard curve formula based on the Ct values of STD1 to STD8. Then, calculate the copy numbers of the tested samples by the standard curve formula. Example for calculating sample concentration is illustrated in Table 4 . The standard curve formula is established by the Ct values and the copy numbers (Log 10 copies/μL) of STD1 to STD8: Y = −3.4167X + 46.917 (R 2 = 0.9988). Y is Ct value acquired by real-time RT-PCR and X is sample concentration presented by copies/μL. UDL is under detection limit

Investigating turkey enteric coronavirus circulating in the Southeastern United States and Arkansas during

Infection with a pathogenic turkey coronavirus isolate negatively affects growth performance and intestinal morphology of young turkey poults in Canada

Detection of turkey coronavirus in commercial turkey poults in Brazil

First full-length sequences of the S gene of European isolates reveal further diversity among turkey coronaviruses

Identification and characterization of a neutralizing-epitopecontaining spike protein fragment in turkey coronavirus

Aromatic amino acids in the juxtamembrane domain of severe acute respiratory syndrome coronavirus spike glycoprotein are important for receptor dependent virus entry and cell-cell fusion

Differential detection of turkey coronavirus, infectious bronchitis virus, and bovine coronavirus by a multiplex polymerase chain reaction

Comparison of virus isolation, immunohistochemistry, and reverse transcriptase polymerase chain reaction procedures for detection of turkey coronavirus

Detection of specific polymerase chain reaction product by utilizing the 50-30 exonuclease activity of Thermus aquaticus DNA polymerase

Specific real time reverse transcription polymerase chain reaction for detection and quantitation of turkey coronavirus RNA in tissues and feces from turkeys infected with turkey coronavirus

The protocol "Real-time reverse transcription-polymerase chain reaction for detection and quantitation of turkey coronavirus RNA in feces and intestine tissues" outlined in this chapter had been successfully carried out in the authors' studies on molecular diagnostics, molecular virology, immunology, and/or vaccinology of turkey coronaviral enteritis. Those studies were in part financially supported by USDA, North Carolina Poultry Federation, and/or Indiana Department of Agriculture and technically assisted by Drs. Tom Brien and David Hermes, Mr. Tom Hooper, and Ms. Donna Schrader for clinical investigation, virus isolation and propagation, and animal experimentation.