J Bacteriol Virol.  2012 Dec;42(4):321-329. 10.4167/jbv.2012.42.4.321.

Isolation and Cloning of an ABC Transporter-Like Gene of Haemophilus parasuis and Its Use in a New Diagnostic PCR

• Kim H
• Cho Y
• Shin S
• Kang S
• Kwon OB
• Hahn TW

• KMID: 2168667
• DOI: http://doi.org/10.4167/jbv.2012.42.4.321

Abstract

The aim of this study was to identify a new gene of Haemophilus parasuis that could be used to develop a polymerase chain reaction (PCR) test for this porcine pathogen. H. parasuis genomic DNA was cloned into a set of expression vectors, and transformants expressing His-tagged polypeptides were identified by colony blotting. An ABC transporter-like gene was isolated. The cloned DNA fragment is 1,105 base pair and shows 78% similarity at the nucleotide level with an ABC transporter gene of H. ducreyi. Based on this sequence, two PCR primers were designed to amplify the entire 1,105-bp fragment in the proposed diagnostic PCR test. PCR amplification was able to detect a minimum of 1 x 10(4) CFU/ml of H. parasuis organisms. Fifteen different H. parasuis serovars were positive using the PCR test. No amplification was observed when the test was done using DNA from 16 other bacterial species commonly isolated from swine.

Keyword

ABC transporter-like gene; Haemophilus parasuis; Diagnostic PCR

MeSH Terms

Base Pairing
Clone Cells
Cloning, Organism
DNA
Haemophilus
Haemophilus parasuis
Peptides
Polymerase Chain Reaction
Swine
DNA
Peptides

Figure

• 

  Figure 1 Agarose gel electrophoresis (1%, v/w) of the plasmid DNA extracted from the putative transformants showing positive reaction against anti-His tag antibody. Lanes 1, molecular weight marker type 100 bp DNA ladder. Lane 2, pET-28b after being linearized with BamH1 restriction enzyme. Lane 3, linearized pET-28b-2A3 inserted plasmid with BamH1 restriction enzyme. Lane 4, pET-28b-2A3 after being digested with restriction enzyme (Not 1-double cut). Lane 5, molecular weight marker type 1 kb DNA ladder.

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  Figure 2 Sequences of 2A3 cloned gene in H. parasuis (Genebank DQ153243) and ABC transporter gene in H. ducreyi 35000HP. Sequence of the gene in H. parasuis is shown in upper lane and those in H. ducreyi is shown in lower lane. When this sequence was compared with database, it showed 78% similarity with ABC transporter, ATPase binding protein, permease gene of H. ducreyi 3500HP strain analyzed by Conserved Domain Database (CDD). The length of cloned gene was 1,105 bp and underlined sequences were the primer sets used in this study.

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  Figure 3 The PCR sensitivity test using DNA extracted from serial dilution of a 1 × 107 CFU/ml H. parasuis culture. Lanes 1, DNA molecular weight marker type 1 kbp DNA ladder; 2, PCR product from 1 × 107 CFU/ml H. parasuis culture; 3, 1 × 106 CFU/ml H. parasuis culture; 4, 1 × 105 CFU/ml H. parasuis culture; 5, 1 × 104 CFU/ml H. parasuis culture; 6~8, showing negative PCR amplification; 9, a positive control for H. parasuis; 10, distilled water as a negative control; 11, DNA molecular weight marker type 100 bp DNA ladder.

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