key: cord-0892028-pjmd8mpv
authors: Wang, X.; Khan, M. I.
title: A multiplex PCR for Massachusetts and Arkansas serotypes of infectious bronchitis virus
date: 1999-02-28
journal: Molecular and Cellular Probes
DOI: 10.1006/mcpr.1998.0204
sha: d697dea68ffa6906dc1068e6e078207f0cf364d7
doc_id: 892028
cord_uid: pjmd8mpv

Abstract Infectious bronchitis virus (IBV), the prototype of the coronavirus family, is an enveloped, single-stranded RNA virus with a genome size of approximately 27·6 kilobase. Infectious bronchitis virus causes an acute, highly contagious respiratory and urogenital disease of chickens which results in significant economic losses in commercial broilers, layers and breeders. A rapid, highly sensitive and specific method is needed in the differential diagnosis of infections of different serotypes. A multiplex polymerase chain reaction (PCR) method was developed and optimized to simultaneously detect Massachusetts (Mass) and Arkansas (Ark) serotypes of IBV. One common primer and two serotype specific primers were chosen from the S1 gene sequences of IBV and used in one PCR reaction. Under optimized PCR conditions, two serotype specific PCR products, 1026 bp for Mass and 896 bp for Ark, respectively, were amplified and detected by agarose gel electrophoreses. The specificity of the technique was verified by using 20 different strains and isolates of IBV, and other avian bacterial and viral pathogens. Using a serial 10-fold dilution of the artificial mixture of both Mass and Ark samples, the detection limit was found to be 5 pg RNA after 35 cycles of PCR. The multiplex PCR was able to detect and differentiate both serotypes in embryonated eggs that were co-infected with different EID50virus titers of Mass 41 and Ark 99. The multiplex PCR developed in this study will be valuable for rapid identification, differential diagnosis, and epidemiological studies of these two serotypes of IBV infections.

Infectious bronchitis virus (IBV), a member of Co-acute, highly contagious respiratory and urogenital disease of chickens that causes significant economic ronaviridae, is a positive sense, single-stranded RNA virus with a genome size about 27·6 kilobase. There losses. 2 The weight gain, feed efficiency, egg production and egg quality are compromised in diseased are three structural proteins encoded in its genome, surface glycoprotein (S), membrane protein (M) and

flocks. In spite of vaccination, there has been an increase in the prevalence of IBV infections due nucleocapsid protein (N). The S protein is cleaved into S1 and S2 during viral maturation. The S1 protein to new serotypes or variant strains, especially in commercial breeders, broilers and layers. 3,4 is responsible for the virus neutralization (VN), haemagglutination inhibition (HI) and serotype specific anti-Diagnosis of IBV infection is accomplished by isolating and serotyping the causative field isolate. The bodies. 1 

DNA from M. gallisepticum S6 was extracted and purified by a previously described method. 15 tests have been used for serotyping. 1, 5 These procedures are expensive, tedious, time-consuming and not widely available to the poultry industry. Serodiagnosis of IBV using serum from recovered flocks Primers selection cannot be used to identify the causative serotypes of IBV because chicken produce cross-reacting anti-Alignment of the S1 gene sequences was performed on the North American IBV serotypes/isolates which bodies following multiple infections. 6 Recently developed molecular methods such as monoclonal include Mass 41, 20 Ark 99, PP14, SE-17, 21 JMK, Gray, 22 Holte (Genebank L18988), Conn 23 and Beaudette, antibodies, 7 polymerase chain reaction (PCR), 8 reverse transcriptase-PCR-restriction fragment length 24 to identify the conserved and variable regions design primers. Serotype specific primers were chosen polymorphism (RT-PCR-RFLP) 9,10 and serotype specific RT-PCR 11 have been used for diagnosis and from a hypervariable region close to the N-terminus of S1 supposed to be associated with serotypes as serotyping IBV infections. While useful, these techniques require multiple steps to identify a particular previously described. 1, 25 One common lower primer was selected from the conserved C-terminus of S1 serotype. Simultaneous detection and differentiation of bacterial, 12-14 mycoplasmal 15 and viral 16 pathogens gene sequences. Primer sequences were analysed for secondary structure formation, G+C content, primer have been described using multiplex PCR amplification techniques.

dimer formation, hairpin formation and their compatibility in the multiplex PCR using the OLIGO The present study describes the development and optimization of a multiplex PCR to detect and dif-primer analysis software and MacDnasis, BESTFIT and BLAST programs 26 (Biotechnology Center, Uni-ferentiate two important serotypes of IBV in a single RT-PCR reaction.

versity of Connecticut, Storrs, CT, USA). The common lower primer is a 24-mer (5′ CGCTCTT AGTA ACA TAAA CTA ACA 3′) sequence located at nucleotide position 1272-1296 in the

Mass 41 20 and 1336-1360 in the Ark 99 21 of S1 gene. The Mass specific upper primer (5′ CTGGGTG Virus and culture conditions TATTGTT GGTACTA TTC 3′) is located at 270-294 nucleotide 20 and the Ark specific primer (5′ ACATTGT Table 1 shows a list of IBV strains representing six TTTAAGA GCGGATCT AA 3′) is located at the 464serotypes and three variants, and other avian patho-488 position 21 of the S1 gene. All primers were syngens and their sources. The IBV and Newcastle Disthesized by the Biotechnology Center, at the ease Virus (NDV) were propagated in 11-day-old University of Connecticut. specific-pathogen-free (SPF) embryonated eggs. The allantoic fluids were harvested after 36 h of incubation at 37°C. Avian reovirus was propagated in chicken Reverse transcription (RT) and multiplex PCR embryo fibroblasts and plaque purified as described reaction previously. 17 Mycoplasma gallisepticum S6 was grown in Frey's media. 18 The reverse transcription reaction was conducted using GeneAmp PCR Kit (Perkin Elmer Cetus, Norwalk, CT, USA). The reaction contained 4 l of 80 m

MgCl 2 , 2 l of 10× PCR buffer [500 m KCl, 200 m Tris HCl (pH 8·4), 0·5 mg ml −1 nuclease-free bovine serum albumin], 2 l of 10 m each dinucleoside Allantoic fluids from embryonated eggs infected with IBV, NDV and cell culture of reovirus were first triphosphate (dNTP), 1 l (20 units) of RNase inhibitor, 1 l (0·5 ) Random Hexamers, 1 l (50 units) of clarified by centrifugation at 500×g for 15 min. Supernatants were transferred to ultracentrifuge tubes.

Moloney Murine Leukemia Virus (MuLV) reverse transcriptase and 50 ng of RNA. A total volume of 20 l The virion pellets were obtained by centrifugation at 93 000×g at 4°C for 3 h. The pellets were then reaction was obtained by adding (DEPC)-treated distilled water. The RT was conducted using a thermal resuspended in 1 ml of diethylpyrocarbonate (DEPC)treated distilled water, followed by the rapid ex-cycler setting of 42°C for 15 min, 99°C for 5 min and 5°C for 5 min for one cycle. Tris HCl (pH 8·4), 0·5 mg ml −1 nuclease-free bovine gels containing 2% agarose (Ultrapure; Bethesda Research Laboratories, Bethesda, MD, USA) with Tris-serum albumin], 1200 ng of common lower primer (500 ng l −1 ), 465 ng of Ark primer (465 ng l −1 ) and borate buffer (45 m Tris-borate, 1 m EDTA). The gel was stained with ethidium bromide (0·5 gm ml −1 ), 347 ng of Mass primer (347 ng l −1 ), 2·5 units of AmpliTaq DNA Polymerase were added in the above exposed to u.v. light to visualize the amplified products, and photographed. RT reaction tubes and 100 l of total volume was obtained by adding DEPC-treated distilled water. Multiplex PCR was performed by 35 cycles of denaturation at 94°C for 1 min, annealing at 63°C for

The specificity and sensitivity of the multiplex PCR 1 min and extension at 72°C for 2 min, followed by final extension at 72°C for 10 min.

To determine the specificity of the technique, 20 different strains of IBV, NDV, Reovirus and M. gallisepticum S6 as listed in Table 1 were examined.

To determine the capability of the multiplex PCR technique to detect and differentiate Mass and Ark Gel electrophoresis was used to detect amplified DNA products. A volume of 15 l of amplified PCR products IBV serotypes in the same reaction, 11-day-old embryonated eggs were inoculated with three different was subjected to electrophoresis at 80 V in horizontal the embryonated eggs (Fig. 3) . From the density of the bands, the relative amounts of Mass and Ark with Ark 99 10 4·2 . Allantoic fluids were harvested after 36 h incubation at 37°C. Total RNA was extracted as virion RNAs can be estimated and are consistent with the titer of each serotype used in the co-infection previously described and 50 ng from each allantoic fluid were used as template RNA. To determine the experiments. Both the VN test and the multiplex PCR technique were used to identify three IBV isolates sensitivity of the multiplex PCR, a serial 10-fold dilution of the artificial mixture of 50 ng of individual from recent outbreaks. The VN test results indicated that two of the isolates were Mass serotypes, one was Mass and Ark were used as template RNAs.

Ark serotype, and the multiplex PCR results agreed with that of the virus neutralization test (data not shown).

The VN test was performed in 10-day-old SPF embryonated eggs as described by Gelb et al. 5 Briefly, DISCUSSION equal parts of a serial of virus dilutions and 10 −1 serum dilution in tryptose phosphate buffer (TPB)

The RT-PCR-RFLP methods described by Kwon et al. 9 and Lin et al. 10 are capable of identifying different were mixed and allowed to react at room temperature for 60 min. Normal serum controls were prepared in serotypes of IBV. However, usually more procedural steps are required to identify the PCR product that TPB at dilution equivalent to the serum concentration used. Embryos inoculated with the serum virus mix-makes such tests somewhat cumbersome and timeconsuming. Most recently, the serotype specific RT-tures were examined for IBV typical lesions (stunting, curling and kidney urates) 7 days post-inoculation.

PCR developed by Keeler et al. 11 greatly improved the speed of serotyping and diagnosis of IBV, but it Reference viruses used were Mass, Ark and JMK. The 50% neutralization end points were calculated as still needs to conduct multiple individual RT-PCR tests to identify a serotype. A Mass and Ark specific described by Reed and Muench. 27 multiplex PCR technique has been developed which has the advantage of rapid identification of multiple serotypes of IBV. It also saves time and money in RESULTS comparison to conducting each individual PCR. Sequence analysis of different serotypes of IBV S1 An IBV specific multiplex PCR amplification technique was optimized which would be able to identify gene suggested that a few amino acids change could give rise to a new serotype, particularly if it occurs Mass and Ark serotypes of IBV in a single RT-PCR reaction. The IBV multiplex PCR products consisted in an area close to the N-terminus of S1 gene. 1, 25 Most of the nucleotide mismatches are concentrated of 1026 bp for IBV-Mass and 896 bp of IBV-Ark serotypes as expected and there were no detectable DNA in the N-terminus of S1 gene, few are found in the C-terminus of S1 gene. Kwok et al. 28 demonstrated bands observed for the other IBV strains or serotypes (Fig. 1) . No spurious PCR amplification reactions that two to three mismatches at the end of 3′ of primers can be specific and can be applied in the between these two serotypes were observed. There were no PCR products generated from the NDV, avian design of strain-specific primers. In this study, one common lower primer was selected from the C-Reovirus and M. gallisepticum S6 nucleic acid (data not shown). The experiment was repeated three times terminus and two serotype specific upper primers from the N-terminus by comparing the known S1 and the results were reproducible. The multiplex PCR assay developed and evaluated in this study was sequences of different serotypes or strains from North America to use in multiplex PCR. found to be a specific assay for IBV Mass and Ark serotypes.

Multiple mismatches at the 3′ end of the Ark upper primer exist when it is aligned with Mass sequence. The multiplex PCR was able to detect RNAs of these two IBV serotypes at levels as low as 5 pg (Fig. There is one mismatch at the 3′ end of Mass primer when it is aligned with the Ark sequence, but eight 2). No spurious PCR amplification between these two serotypes were observed using various amounts of out of the 24 nucleotides are different. Initially, a low annealing temperature 50°C was used. All four strains both RNA mixtures. In addition, the amounts of the amplified products showed a linear correlation to the of Mass serotype yielded a strong specific PCR product corresponding to 1026 bp. All three strains of Ark amount of RNAs in the dilution. Using the multiplex PCR, both IBV-Mass and IBV-Ark were detected in serotype generated specific PCR product about processing environment and in poultry products and their confirmation by multiplex PCR. Applied and Environmental Microbiology 60, 4600-4. 14. Kulski, J. K., Khinsoe, C., Pryce, T. & Christiansen, K.

Pathogenicity for chickens of a reovirus pp

Infectious bronisolated from turkeys

Iowa State A medium for the isolation of avian mycoplasmas

Single-step method of RNA isolation by acid guanidinium thiserotypes of infectious bronchitis virus: their role in disease

Variant 20

Experimental confirmation of recombination upstream The peplomer protein sequence of the M41 strain of coronavirus IBV and its comparison with Beaudette of S1 hypervariable region of IBV

Journal of dence of natural recombination within the S1 gene of infectious bronchitis virus

Molecular cloning and sequence comparison of the S1 glyestimating fifty percent end points

Evolutionary implications of genetic of primer-template mismatches on the polymerase chain reaction: Human immunodeficiency virus type variations in the S1 gene of infectious bronchitis virus

Epidemiology of infectious bronchitis in the United States. In Interquences of coronavirus IBV strains M41 and 6/82 with that of IBV Beaudette

 (1995). Use of multiplex PCR to detect and identify Mycobacterium avium and M. intercellulare in blood