A universal method for the identification of bacteria based on general PCR primers

Abstract

The Universal Method (UM) described here will allow the detection of any bacterial rDNA leading to the identification of that bacterium. The method should allow prompt and accurate identification of bacteria. The principle of the method is simple; when a pure PCR product of the 16S gene is obtained, sequenced, and aligned against bacterial DNA data base, then the bacterium can be identified. Confirmation of identity may follow. In this work, several general 16S primers were designed, mixed and applied successfully against 101 different bacterial isolates. One mixture, the Golden mixture7 (G7) detected all tested isolates (67/67). Other golden mixtures; G11, G10, G12, and G5 were useful as well. The overall sensitivity of the UM was 100% since all 101 isolates were detected yielding intended PCR amplicons. A selected PCR band from each of 40 isolates was sequenced and the bacterium identified to species or genus level using BLAST. The results of the UM were consistent with bacterial identities as validated with other identification methods; cultural, API 20E, API 20NE, or genera and species specific PCR primers. Bacteria identified in the study, covered 34 species distributed among 24 genera. The UM should allow the identification of species, genus, novel species or genera, variations within species, and detection of bacterial DNA in otherwise sterile samples such as blood, cerebrospinal fluid, manufactured products, medical supplies, cosmetics, and other samples. Applicability of the method to identifying members of bacterial communities is discussed. The approach itself can be applied to other taxa such as protists and nematodes.

Keywords: Bacterial identification; Golden mixture; Multiplex; Universal method; Universal primers.

Fig. 1

A flowchart presentation of the UM protocol; starting from pure bacterial culture or DNA, ending with DNA alignment (BLAST) and bacterial identification

Fig. 2

All nine Level reactions (Table 5A) were positive with isolate QUBC26. Lanes represent Level reactions in the same order (1–9). Interpreted results are shown in the table

Fig. 3

Reaction 2b (lanes1–5) and Reaction 3 (lanes 8–12 (Table 5A). Lanes 1 and 8 QUBC43 Shigella (API), lanes 2 and 9 QUBC42 Salmonella (API), lanes 3 and 10 QUBC41 Klebsiella (API), lanes 4 and 11 QUBC40 Proteus, and lanes 5 and 12 QUBC34 Staphylococcus aureus. Notice the presence of the 510 bp in lanes3–5 and its absence from lanes 1 and 2 and the presence of a 630 bp band in lanes 8 and 9 and its absence in lanes 10, 11, and 12

Fig. 4

G4 has failed to detect this bacterium; Streptomyces sp.(QUBC50), notice the absence of the 630 bp which appeared with G2, G4 showed very week band at 287/298 bp that was clearly present with G3

Fig. 5

a (QUBC56) and b (QUBC58) tested with different Golden mixtures; G4–G12, showing poor performance of G8 (asterisks)

Fig. 6

Detection of bacterial rDNA from 12 different QUBC isolates with G7 (a) or with G10 (b), notice that isolate QUBC4 was negative with G10

Fig. 7

Ten-fold serially diluted DNA samples from clinical Escherichia coli isolate QUBC62; API 20E, were tested with G7 (a) or G10 (b). 100-bp ladder markers were included