Chapter 3 : Laboratory Methods Used for Strain Typing of Pathogens: PCR-Based Strain-Typing Methods

PCR. The entire reaction is carried out in a microfuge tube (100-μl volume) that contains all of the reagents needed to amplify a segment of the DNA molecule of interest. When the tube containing a double-stranded DNA fragment is heated to about 95°C, it separates into single strands (denaturation) (1). In the second step (2), the temperature of the reaction mixture is lowered to about 40 to 60°C, which allows synthetic pieces of oligonucleotide (primers) to bind (anneal) to its complementary sequence (template). Then, at 70 to 75°C, the enzyme polymerase adds nucleotides along the template DNA away from the primer binding site (extension) (3). At each cycle of this three-step process, the target DNA fragment doubles in number (2x). Hence, the only varying condition that is applied to the reaction mixture is the temperature, which is regulated by a device called a thermocycler. (Illustration by Ariana Reynolds.)

AFLP. Chromosomal DNA is digested with two different restriction endonucleases, which generate DNA fragments with distinct overhangs at the ends. These ends are then ligated (with an enzyme DNA ligase) to synthetic linkers with known nucleotide sequences, which serve as templates for PCR primers. In this way, all DNA fragments ligated to the linkers are amplified, if the length of the fragment is short enough. The amplified fragments are then resolved by gel (usually acrylamide) electrophoresis. The resolved bands generate a fingerprint pattern, which can then be compared among different strains. (Illustration by Ariana Reynolds.)

RSS-PCR gel electrophoresis. A segment of E. coli chuA gene that encodes an outer heme receptor protein was amplified by a set of primers designed to have a mismatch at the 3′ end of a restriction site in the target DNA sequence. This procedure was able to differentiate E. coli O157:H7 isolates (lanes 2 and 3) from E. coli isolates belonging to enteropathogenic E. coli serotype O111: NM (lane 5), enterotoxigenic E. coli strain H10407 (lane 6), enteroaggregative E. coli strain 25-2 (lane 7), and enteroinvasive E. coli strain 11 (lane 8). Lanes 9 and 10 are Salmonella enterica serotype Enteritidis strains, phage type 4 and 8, respectively. Lane 4 is an atypical enteropathogenic E. coli serotype O55:H7, shown by other strain-typing methods to be closely related to the E. coli serotype O157:H7 lineage (see text). Lane 1 is an MW marker ladder.

Repetitive element PCR. In this PCR-based strain-typing procedure, DNA sequences between repetitive DNA elements are amplified by primers designed to be extended away (outward) from the repetitive element sequences. Thus, multiple amplified fragments are generated, depending on the sequence length between the repetitive elements. These multiple fragments will then generate a fingerprint pattern upon gel electrophoresis. The figure shows segments of 600 to 6,000 bp that could be potentially amplified from the 10,000-bp target by primer 1 and primer 2. The length of DNA segment that can be amplified depends on the quality of the polymerase used, extension time, and reagent conditions in the PCR mixture. (Illustration by Ariana Reynolds.)

Spoligotyping of M. tuberculosis DNA. (A) The M. tuberculosis genome contains multiple copies of a 36-bp sequence interspersed with unique spacer sequences (DRs) that are 34 to 41 bp long. (B) Synthetically constructed oligonucleotides based on the spacer regions of a reference M. tuberculosis strain are immobilized on a membrane and serve as targets of a hybridization reaction. The hybridization probes are constructed by PCR with primers DRa and DRb, which are designed to amplify spaces between the DR loci of a test M. tuberculosis strain (1). The amplified products are then applied (2) into slit wells of a hybridization manifold containing the membrane (multiple arrows). (C) The probes that bind (hybridize) to specific spacers in the membrane are then visualized (3). Black spots shown in rows indicate spacer sequences present in a particular test strain that are also present in the reference strain. The patterns of these spots are then compared. (Adapted by Laura Flores from reference 26, with permission.)