Detection of Fish Pathogens Using Variable Number Tandem Repeat (VNTR) Analysis

Abstract

Source: Gulla, S., et al. Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis. J. Vis. Exp. (2019).

This video demonstrates the procedure for separating fluorescently labeled variable number tandem repeat (VNTR) amplicons, derived from a fish-pathogenic bacterium, using capillary electrophoresis to generate size-specific electropherogram profiles for bacterial identification.

Protocol

1. Capillary Electrophoresis Setup and Run Conditions

1. Following confirmation of PCR (Polymerase chain reaction) amplicons, dilute PCR products 1:10 (v/v) in purified water. Seal, mix, and centrifuge briefly.
2. Working in a fume cupboard, prepare a volume of master mix consisting of 9 µL of formamide and 0.5 µL of size standard per PCR product (allow 10% surplus volume). Vortex briefly and distribute 9.5 µL into wells on a plate appropriate for the available CE (Capillary electrophoresis) system, before adding 0.5 µL of diluted PCR product. Seal, mix, and centrifuge briefly.
   CAUTION: Handle with care. Mixing formamide with water ge....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Avant 3500xL Genetic Analyzer	Thermo Fisher Scientific	A30469	Used for capillary electrophoresis fragment analysis.
BioNumerics7 modules	Applied Maths	NA	Used for generating Minimum spanning trees from MLVA data.
Centrifuge(s)	As preferred.	NA
Eppendorf Safe-Lock Tubes, 1.5 mL	Eppendorf	30120086	Centrifuge tubes used during DNA extraction.
Freezer	As preferred.	NA
Fume cupboard	As preferred.	NA
GeneMapper Software 5	Thermo Fisher Scientific	4475073	Used for reading electropherograms from capillary electrophoresis.
GeneScan 600 LIZ dye Size Standard v2.0	Thermo Fisher Scientific	4408399	Size standard used during capillary electrophoresis.
Hi-Di Formamide	Thermo Fisher Scientific	4311320/4440753	Deionized formamide used during capillary electrophoresis. Prepare working aliquouts.
Milli-Q water	NA	NA	Purified water used during PCR and capillary electrophoresis. Standard recipe; produced in-house.
Multiplex PCR Plus Kit	Qiagen	206151/206152
PCR thermal cycler	As preferred.	NA
POP-7 Polymer for 3500 Dx/3500xL Dx Genetic Analyzers	Thermo Fisher Scientific	A26077/4393713/4393709	Separation matrix used during capillary electrophoresis.
Pure culture Yersinia ruckeri	NA	NA	E.g. cryopreserved or fresh.
RNase-free water	Qiagen	NA	In: Multiplex PCR Plus Kit
Tris-borate-EDTA (TBE) buffer	NA	NA	Standard recipe; produced in-house.
Trypsine soy agar/bovine blood agar	NA	NA	Standard recipe; produced in-house.
Vortexer	As preferred.	NA