Once the serogroups were identified, the results of a second round of reactions that used primers and probes for the flagellar antigen l genes, 1,2; e,h; g,m; d; e,n,x; and z10, and the Vi gene were used to identify individual serovars.
Firstly, an initial screen of the quality of the samples was performed from the first round of PCR reactions [14].
This solution was then diluted 1 in 10 in sterile deionized, distilled water, and 5 µL was included in the first round of PCR reactions.
In a first round of PCR reactions, a 1549 bp backbone fragment from pPZP200 was amplified using primers PZPN-1/Kpn2Ifor and PZPN-1rev (see Table 1 for primers and Table 2 for vectors used in this work) and a 1283 bp fragment was amplified by using primers PZPN-2for and PZPN-2/XhoIrev.
The first round of PCR reaction with the outer primer pair sets (M022/M338 for pBER and pBDR insertion, and "M022/M026" for pBGV and pBGW insertion) was performed using TaKaRa LA-Taq (#RR002A; TaKaRa) in a total of 10 μl solution.
By using the sense strand ssDNA of one gene and the antisense-strand ssDNA of another gene as starting materials for DNA family shuffling, high recombination efficiency can be expected, as only heteroduplex molecules are formed in the first round of polymerase chain reaction (PCR).
In the first round of amplifications, each reaction used 200 ng of DNA template, 2.5 mM MgCl2, 10 µM kDNA primer (S34 or S67), 0.2 mM dNTPs, 2.5 U Taq Platinum (Invitrogen, Carlsbad, CA).
Reverse transcription and the first round of PCR (25-μl reactions) were performed using One-Step RT-PCR with Platinum Taq (Invitrogen).
At the end of the first round of asymmetric PCR, the two reactions were combined and allowed to proceed through 20 more PCR amplification cycles.
