Detection of Saccharibacteria in Binary Culture Using Polymerase Chain Reaction

Samenvatting

Source: Collins, A. J., et al., Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity. J. Vis. Exp. (2021)

This video demonstrates the use of Polymerase Chain Reaction (PCR) to detect Saccharibacteria in a binary culture with Actinomyces. The protocol outlines DNA amplification and gel electrophoresis to confirm a stable host-parasite interaction.

Protocol

1. Confirm Saccharibacteria infection by PCR
   1. After 5 passages, confirm infection with the polymerase chain reaction (PCR). Five passages will ensure that any non-growing Saccharibacteria cells have been depleted beyond the limit of detection using 25 cycles of PCR.
   2. Prepare a PCR master mix. A suggested recipe is as follows: for each tube needed, prepare 12.5 µL 2x PCR buffer, 0.75 µL 10 µM Forward primer (580F¹² - AYT GGG CGT AAA GAG TTG C), 0.75 µL 10 µM Reverse primer (1177R¹³- GAC CTG ACA TCA TCC CCT CCT TCC), 1 µL 25 mM MgCl_2, and 9 µL water. Mix by vortexing.
   3. Aliquot 24 µL of master mix into a 0.2 mL PCR tube. Add 1 µL of Saccharibacteria-infected culture to the PCR reaction. Place the tube in a thermocycler and perform the following protocol: Initial denaturation at 95 °C for 5 min, 25 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, final elongation at 72 °C for 2 min, then final hold at 4 °C.
   4. Load and run the PCR products in a 1% agarose gel. A band of ~600 bases will indicate the presence of Saccharibacteria.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Agarose	Fisher Scientific	BP160-100
Brain Heart Infusion Broth (dehydrated powder)	Becton-Dickinson	211059	Or other growth media suitable for target organisms
Electrophoresis Power Supply	Bio-Rad	1645052
Electrophoresis Rig	Bio-Rad	1704467
GoTaq Green Mastermix	Promega	M7122
Mastercycler Pro Thermocycler	Eppendorf	950040025	Or equivalent thermocycler for PCR
MgCl₂ solution 25mM	Promega	A3513
Molecular Biology grade water	Fisher Scientific	BP2819100
P-1000 micro pipette	Gilson	F123601G
PCR tubes 0.2 mL	Fisher Scientific	14-230-205
Pipette tips - 1000 μL	Fisher Scientific	02-717-166
SYBR Safe DNA Gel stain	ThermoFisher Scientific	S33102
Tryptic Soy Broth (dehydrated powder)	Becton-Dickinson	211825	Or other growth media suitable for target organisms