**4.4 Primers and PCRs used to amplify P- and F17-fimbrial genes**


Primers used in the PCRs to amplify the studied genes are listed in **Table 2**.

#### **Table 2.**

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**Dog's age Clinical signs Serotype MSHA MRHA**

 Unknown Unknown O78K? E, P, C C Young dog Chronic diarrhoea O6K53 E, P, C, O P, O Unknown Unknown NT B, E, P, C, O B, E, P, O Pup Sepsis O20K? None P Unknown Unknown O6K- B, E, P, C, O P, C, O Pup Sepsis diarrhoea O4K2 B, P, C, O E, P, C, O Pup Sepsis diarrhoea O42K- E, P, C None 5 months Chronic diarrhoea O6K1 E, P, C, O P, C Pup Sepsis diarrhoea O23K1 B, E, P, C P, C, O Unknown Unknown O42K- C, O None Pup Sepsis diarrhoea O4K53 E, P, C, O O 3 months Recurrent diarrhoea O4K? E, P, C, O P, C, O 3 months Unknown O139K? E, P, C P 18 months Chronic diarrhoea O4K? E, P, C, O E, P, C, O 4 years Chronic diarrhoea O25K13 E, P, C, O P, C, O 12 months Chronic diarrhoea O4K? P, O P, O 9 months Chronic diarrhoea NT P, C, O P, O Unknown Chronic diarrhoea O6K53 B, E, P, C, O B, E, P, C, O Old dog Chronic diarrhoea O4K- E, P, C, O E, P, C, O Pup Sepsis diarrhoea NT B, C, O None Unknown Chronic diarrhoea O4K? B, E, P, C B, E, P, C 4 months Unknown O4K? E, P, C, O O Unknown Chronic diarrhoea O4K- E, P, C, O E, P, C, O 3 months Bloody diarrhoea O2K1 B, E, P, C, O B, E, P, C, O *MSHA is an abbreviation of mannose-sensitive haemagglutination, and MRHA is an abbreviation of mannoseresistant haemagglutination. The erythrocytes are abbreviated as follows: B, bovine erythrocytes; E, equine* 

**Strain number**

DNA in 70% ethanol was pelleted with centrifugation (10 min at 14,000 rpm at room temperature). The 70% ethanol was then removed and the chromosomal DNA pellet air-dried at 37°C. Finally the chromosomal DNA pellet was dissolved in

One μl of the isolated chromosomal DNA was used in 50 μl PCR mixtures consisting of 20 pmol of each primer, 0.2 mM dNTP mixture and 0.625 U of *Taq*-polymerase in PCR buffer [5]. In PCRs for P-fimbrial genes for positive control samples, the isolated chromosomal DNA of the dog uropathogenic *E. coli* strain (strain 1473) was used. In PCRs for F17-fimbrial gene for positive control samples, the isolated chromosomal DNA of the cattle mastitis *E. coli* strain (strain E5) was used. In all PCRs for the negative control, sterile distilled water was used [31].

**4.3 PCR mixtures for PCR amplification of P- and F17-fimbrial genes**

**170**

**Table 1.**

100 μl of sterile distilled water.

*erythrocytes; C, canine; O, ovine; P, porcine. NT, non-typable.*

*Characteristics of the 24 studied E. coli strains [5, 31].*

*Primers and their melting temperatures (Tm) used to amplify the studied genes.*

Predicted primer annealing sites of the used primers on the target operons are shown in **Figure 2**.

The PCR amplification in all the reactions for all studied genes was carried out in the following steps: heating at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min and the final extension for 10 min at 72°C.

The expected sizes of PCR products were determined with the 'Primer-BLAST' online tool (data set nr organism *Escherichia coli*) on the Internet page of the National Center for Biotechnology Information, US National Library of Medicine (http://www. ncbi.nlm.nih.gov) as follows: *papA*—552 bp (GenBank deposited nucleotide sequence LR134092.1), 555 bp (GenBank deposited nucleotide sequence CP025703.1), 534 bp (GenBank deposited nucleotide sequence CP018957.1), 564 bp (GenBank deposited nucleotide sequence CP029579.1) and 561 bp (GenBank deposited nucleotide sequence CP024886.1); *papEF*—1372 bp (GenBank deposited nucleotide sequence CP027701.1), 1373 bp (GenBank deposited nucleotide sequence CP028304.1), 1367 bp (GenBank deposited nucleotide sequence CP026853.1) and 1371 bp (GenBank deposited nucleotide sequence LR134238.1); *papG*—1000 bp (GenBank deposited nucleotide sequence CP026853.1) and 1003 bp (GenBank deposited nucleotide sequence M20181.1); and *F17G*—888 bp (GenBank deposited nucleotide sequence AF055313.1) and 885 bp (GenBank deposited nucleotide sequence CP001162.1).

## **4.5 Agarose gel electrophoresis**

Samples of isolated chromosomal DNA (5 μl of isolated chromosomal DNA and 1 μl of 6 × loading dye) were subjected to analysis with agarose gel electrophoresis using

1% of agarose gels with 0.5 μg/ml ethidium bromide, run in 0.5 × TBE electrophoresis buffer. Samples of obtained PCR products (25 μl of PCR products, 5 μl of 6 × loading dye) were subjected to analysis with agarose gel electrophoresis using 1% of agarose gels with 0.5 μg/ml ethidium bromide, run in 1 × TAE electrophoresis buffer. Used protocols for agarose gel electrophoresis were based on Sambrook et al. [34]. For DNA ladder the lambda bacteriophage DNA digested with the restriction endonuclease *Pst*I was used.
