Development of a Multiplex PCR and PCR-RFLP Method for Serotyping of Avibacterium paragallinarum

Abstract

ABSTRACT. Avibacterium (Haemophilus) paragallinarum (A. paragallinarum) is a causative agent of infectious coryza in chickens and is classified into three serovars by agglutination tests. In an effort to identify the serovars easily, PCR and PCR-RFLP were employed. As the target gene for PCR, the hypervariable region of HMTp210, which encodes the HA antigen, was used. PCR using primer sets around the hypervariable region amplified 0.8, 1.1 and 1.6 kbp fragments for serovars A, B and C, respectively. Alternatively, the 1.6 kbp fragments were amplified with another primer pair encompassing the hypervariable region and was subjected to digestion with Bgl II, which resulted in the detection of serovar-specific digestion patterns. These results indicate that PCR and PCR-RFLP using the hypervariable region of HMTp210 are alternative methods to identify the serovar of A. paragallinarum.

An evaluation of serotyping of Avibacterium paragallinarum by use of a multiplex polymerase chain reaction

“…17 It is known that a region within the HMTp210 gene (termed region 2), shows, on the basis of a limited data set, sequence diversity between Page serogroups, a diversity that has been exploited in the proposed PCR. 15 In the current study, this proposed PCR alternative to conventional serotyping has been evaluated with the full set of Page and Kume reference strains as well a number of diverse Av. paragallinarum field isolates.…”

“…12 All reference strains and field isolates included in the study were examined by a serotyping mPCR as reported. 15 Briefly, the genomic DNA was extracted by using a commercial kit, a according to the manufacturer's instructions. The PCR reactions were performed in a total volume of 25 μl containing 2.5 μl of 10× PCR buffer included in the kit, b 0.2 mM of each deoxyribonucleotide triphosphate, 0.2 μM of each Page serovar-specific primer: ABC forward 5′-GGCTCACAGCTTTATGCAA CGAA-3′; A reverse 5′-CGCGGGATTGTTGATTTTGT T-3′; B reverse 5′-GGTGAATTTCACCACACCAC-3′; and C reverse 5′-TAATTTTCTTATTCCCAGCATCAATA CCAT-3′.…”

“…The PCR reactions were performed in a total volume of 25 μl containing 2.5 μl of 10× PCR buffer included in the kit, b 0.2 mM of each deoxyribonucleotide triphosphate, 0.2 μM of each Page serovar-specific primer: ABC forward 5′-GGCTCACAGCTTTATGCAA CGAA-3′; A reverse 5′-CGCGGGATTGTTGATTTTGT T-3′; B reverse 5′-GGTGAATTTCACCACACCAC-3′; and C reverse 5′-TAATTTTCTTATTCCCAGCATCAATA CCAT-3′. 15 A total of 1.25 units of Taq DNA polymerase b and 2 μl of extracted DNA were used. The amplification steps were as reported previously: 98°C for 1 min; 30 cycles of 98°C for 10 sec, 56°C for 10 sec and 72°C for 2 min, and a final step at 72°C for 7 min.…”

“…The amplification steps were as reported previously: 98°C for 1 min; 30 cycles of 98°C for 10 sec, 56°C for 10 sec and 72°C for 2 min, and a final step at 72°C for 7 min. 15 The PCR products were subjected to agarose gel electrophoresis using 2% agarose, visualized, and photographed under ultraviolet light. Multiplex PCR serotyping of all isolates and reference strains included in the study were performed in both testing laboratories (Mexico and Peru).…”

“…Reference strains 221, 0083, 0222, and Modesto were correctly identified as serogroup A, A, B, and C, respectively, as previously reported. 15 Reference strain 2671 (serogroup B) was identified as serogroup B. However, reference strains 2403, E-3C, and HP14 of serogroup A were misidentified as serogroup B.…”