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

Abstract

Turkey coronavirus (TCoV) infection causes acute atrophic enteritis in the turkey poults, leading to significant economic loss in the U.S. 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 (RRT-PCR) assay for detection and quantitation of TCoV in the turkey tissues was developed using a dual-labeled fluorescent probe. The fluorogenic probe labeled with a reporter dye (FAM, 6-carboxytetramethylrhodamin) and a quencher dye (AbsoluteQuencher) was designed to bind to a 186 base-pair fragment flanked by the two PCR primers targeting the 3' end of spike gene of TCoV. The assay was performed on different avian viruses and bacteria to determine the specificity as well as serial dilutions of TCoV for the sensitivity. Three animal trials were conducted to further validate the assay. Ten-day-old turkey poults were inoculated orally with 100 EID(50) of TCoV. Intestinal tissues (duodenum, jejunum, ileum, cecum), feces from the cloacal swabs, or feces from the floor were collected at 12 h, 1, 2, 3, 5, 7, and/or 14 days post-inoculation (DPI). RNA was extracted from each sample and subjected to the RRT-PCR. The designed primers and probe were specific for TCoV. Other non-TCoV avian viruses and bacteria were not amplified by RRT-PCR. The assay was highly sensitive and could quantitate between 10(2) and 10(10) copies/microl of viral genome. The viral RNA in the intestine segments reached the highest level, 6x10(15) copies/microl, in the jejunum at 5 DPI. Eighty-four intestine segments assayed by the developed RRT-PCR and immunofluorescence antibody assay (IFA) revealed that there were 6 segments negative for TCoV by both assays, 45 positive for TCoV by IFA, and 77 positive for TCoV by RRT-PCR. Turkey coronavirus was detected in the feces from the cloacal swabs or floor 1-14 DPI; however, the viral RNA load varied among different turkey poults at different intervals from different trials. The highest amount of viral RNA, 2.8x10(10) copies/microl, in the feces was the one from the cloacal swab collected at 1 DPI. The average amount of TCoV RNA in the cloacal fecal samples was 10 times higher than that in the fecal droppings on the floor. Taken together, the results indicated that the developed RRT-PCR assay is rapid, sensitive, and specific for detection, differentiation, and quantitation of TCoV in the turkey tissues and should be helpful in monitoring the progression of TCoV induced acute enteritis in the turkey flocks.

Fig. 1

Specificity of real-time RT-PCR (RRT-PCR) for turkey coronavirus (TCoV). (A) The products of RRT-PCR with the primers QS1F and QS1R and the probe QS1P performed on RNA extracted from TCoV isolate 517, 540, ATCC, 310, 1440, and 428 are shown in 1% agarose gel from lanes 3 to 8, respectively. Lane 1 is DNA marker 100-base pair (bp) (Invitrogen, Carlsbad, CA, USA) and lane 2 is RNA in vitro transcribed from plasmid pTriEx3-6F/6R as the positive control. (B) The products of RRT-PCR performed on RNA extracted from TCoV isolate 540, canine coronavirus (CCoV), bovine coronavirus (BCoV), transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus (FIPV), infectious bronchitis virus (IBV) Ark99, IBV Conn, and IBV M41 are shown on 1% agarose gel from lanes 2 to 9, respectively. Lane 1 is 100-bp DNA marker. (C) The products of RRT-PCR performed on RNA extracted from Salmonella, Escherichia coli, avian influenza virus, Newcastle disease virus, avian reovirus, turkey herpesvirus, enterovirus, and rotavirus are shown on 1% agarose gel from lanes 1 to 8, respectively. Lane 9 is 100-bp DNA marker and lane 10 is TCoV 540 as the positive control.

Fig. 2

Standard curve of real-time RT-PCR (RRT-PCR) assay for turkey coronavirus (TCoV). The X-axis shows the concentration of serially diluted RNA in vitro transcribed from standard pTriEx3-6F/6R in log 10 value and the Y-axis indicates the corresponding threshold cycle (Ct) value. The linear quantitative correlation was established from 10² to 10¹⁰ copies/μl with 99.8% coefficient. The detection limit is as low as 10² copies/μl.

Fig. 3

Detection of turkey coronavirus (TCoV) in the intestinal tissue samples from experimentally infected turkeys in the trial 1. The results by immunofluorescent antibody assay (IFA) are compared to those by real-time RT-PCR (RRT-PCR). The numbers indicate the numbers of samples positive (+) or negative (−) for TCoV.

Fig. 4

The turkey coronavirus (TCoV) levels in the intestinal tissue samples from experimentally infected turkeys determined by real-time RT-PCR (RRT-PCR) in the trial 1. Different intestinal segments are indicated by different columns. The TCoV level is calculated and shown as copies/μl. The bar in each column is standard deviation. Asterisks indicate significantly differences (p < 0.05) found among different intestinal segments collected at the same time point.

Fig. 5

The concentration of turkey coronavirus (TCoV) in the cloacal fecal swab samples collected sequentially after inoculation from the same three turkeys for 14 days in the trial 2. Three lines with different shapes (♦, ■, ▴) represent three different turkeys (individual number 102, 103, and 104). The TCoV level is calculated and shown as copies/μl.

Fig. 6

The concentration of turkey coronavirus (TCoV) in the fecal droppings on the floor assayed by real-time RT-PCR (RRT-PCR) from 1 to 14 days after inoculation in the trial 3. The blue line with shape (♦) represents samples of TCoV-infected turkeys and the pink line with shape (■) represents samples of the negative control group. The TCoV level is calculated and shown as copies/μl. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)