Duplex PCR To Differentiate between Mycoplasma synoviae and Mycoplasma gallisepticum on the Basis of Conserved Species-Specific Sequences of Their Hemagglutinin Genes

Abstract

We developed a duplex PCR assay targeting the hemagglutinin multigene families, vlhA and pMGA, of Mycoplasma synoviae and Mycoplasma gallisepticum, respectively. The assay proved to be specific and sensitive enough to justify its use for the simultaneous detection of the two major avian mycoplasma species from field isolates.Mycoplasma gallisepticum and M. synoviae are considered important in commercial poultry industries. M. gallisepticum causes chronic respiratory disease in chickens and sinusitis in turkeys (17), and M. synoviae is commonly involved in respiratory tract infection, synovitis in chickens, and poor growth (12). The antigenic relatedness (1, 2, 7, 11) of these organisms make them difficult to identify with conventional serological tests (5, 6). Attempts to differentiate between these two major avian mycoplasmas by using molecular methods, such as PCR tests, were mainly based on the 16S rRNA gene (4, 10, 13). However, it often requires additional steps, such as restriction fragment length analysis (9) or hybridization with species-specific probes (3, 10). In addition, these analyses resulted in concomitant amplification of unrelated bacterial DNA, making this approach useless for the testing of clinical material.We show here that PCR amplification targeting speciesspecific structural genes provides an efficient tool for the simultaneous detection and differentiation of M. gallisepticum and M. synoviae.The Mycoplasma species and walled bacteria used in the present study are listed in Table 1. All of the mycoplasma strains were propagated in Frey's medium (8). Unrelated bacterial species were cultured in brain heart infusion broth (Difco Laboratories, Detroit, Mich.). Forty tracheal swabs were immediately processed for culture and used as clinical mycoplasma samples to test the applicability of the duplex PCR assay.The template DNA of M. synoviae and M. gallisepticum was prepared from 200 l of culture, to which an equal volume of nonionic detergent mix solution (0.45% Nonidet P-40, 0.45% Tween 20, and 100 g of proteinase K/ml) was added. The sample was incubated at 56°C for 1 h, boiled for 10 min, and then centrifuged at 14,000 ϫ g for 5 min. Then, 10 l of the resulting supernatant was directly used for PCR.All of the primers used in the present study are listed in Table 2. They targeted pMGA1.2 gene encoding a hemagglutinin protein (pMGA) from M. gallisepticum (14) and M. synoviae2/12 DNA fragment (accession no. M. synoviaeU66314) (3) of the M. synoviae hemagglutinin vlhA gene (15, 16). To confirm the specificity of the amplification reaction, a nested PCR was performed by using inner primers (Tab.2). Additional avian Mycoplasma spp. and other common bacteria (Table 1) were tested to determine the specificity of the duplex PCR assay.The amplification reaction was performed in a total volume of 50 l containing 5 l of 10ϫ PCR buffer (Amersham Biosciences), 250 M concentrations of each deoxynucleoside triphosphate (Pharmacia), 50 pmol of each external or internal primers, 2.25 mM MgCl 2 , 2 U of Taq D...

Isolation and Identification of Mycoplasma synoviae from Suspected Ostriches by Polymerase Chain Reaction, in Kerman Province, Iran

“…To control the bacterial and fungal contamination of samples, resistant antibiotic resistance as well as filtration should be considered. Microbiological method is needed for some research projects and even for diagnosis; to prepare the PCR samples, many false negative PCR results might occur without enrichment ( 19 ); therefore, culturing should not be ignored. But culturing can be costly and time-consuming, and can also be inconclusive because of low sensitivity ( 20 ).…”

Comparison of three diagnostics methods of Mycoplasma gallisepticum in Batna governorate (Algeria)

“…1: PCR results for 11 MG strains (well 1 is molecular weight marker). The discordance observed between culture and PCR may be attributed to different factors such as: the addition of a greater quantities of antibiotics were ineffective in controlling bacterial contamination during strains transport to France (OIE Terrestrial Manual, 2008) ; during transport, refrigeration of samples should be considered because the Mycoplasma die quickly at room temperature (Pourbakhsh et al, 2010); The possibility of losing some atypical strains of Mycoplasma by PCR technique especially if they have experienced multiple passages on artificial medium (Ben Abdelmoumen Mardassi et al, 2005) ; The antigenic community between Mycoplasma species particularly MG and MS can explain an isolation rate more important (Ben Abdelmoumen, 1996).…”

“…It can yield positive results more frequently than culture. The main disadvantage to PCR is its ability to amplify the DNA of all Mycoplasma (viable or non-viable) who could stay in the body of animals giving false positive reactions (Aimeur et al, 2010) and although the culture is long and tedious, it must be done in parallel to PCR to provide more security for diagnosis (Ben Abdelmoumen Mardassi et al, 2005).…”

Molecular identification of Mycoplasma synoviae from seroprevalent commercial breeder farms at Chittagong district, Bangladesh

“…Earlier MS-specific PCRs were based on the 16S rRNA gene [44,45], and more recently, some have been based on hemagglutinin genes [15,46]. In this study, species-specific primers of Pérez et al .…”