Part:BBa_K3416102

Introduction

Vilnius-Lithuania iGEM 2020 project FlavoFlowincludes three goals towards looking for Flavobacterium disease-related problems solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and creating a foundation of edible vaccines. This part was used for the first goal- detection - of the project FlavoFlow.

Overview

Vilnius Lithuania iGEM 2020 team decided to create a lateral flow assay (LFA) test for Flavobacterium identification and detection purposes. F. columnare causes columnaris disease in freshwater fish. The infection results in skin and fin erosions as well as gill necrosis and eventually leads to death¹. It is essential to detect the infection-causing pathogen as soon as possible so that an appropriate treatment could be started. To do this, our team created a helicase dependent amplification (HDA)-LFA based detection test that in a few hours can identify an exact bacteria.

Detection system

Lateral flow assay based on nucleic acid requires three single-stranded DNA probes: detection, capture, and control. The main principle of this method is that the added ssDNA amplicon hybridizes to the detection probe as well as capture probe, due to this first visible red line appears, eventually a second line also appears due to the hybridization of control and detection probe. If two lines are present, then the test is positive, if only one is visible - negative.

Bioinformatic analysis

Usually, for phylogenetic analysis and identification 16S rRNA gene can be used². For this reason, we developed LFA probes based on this gene sequence. F. columnare 16S rRNA gene (AY577821) was chosen as a marker sequence. To make sure that the LFA test is highly specific, we made a multiple sequence alignment with 16S rRNA genes from other species within the same genus using Clustal Omega tool (1. 2. 4.). Unique target sequences for F. columnare LFA probes were selected based on the absence of matching alignments between sequences (Fig. 1).

Figure 1. 1 - Flavobacterium species 16S rRNA partial gene sequences alignment. Black boxes highlight sequence parts chosen for probe placement. Gene sequences: F. columnare - AY577821, F. branchiophilum - AB680752, F. psychrophilum - AY662493.

To develop the F. columnare LFA test based on 16S rRNA gene these parts are needed: BBa_K3416101, BBa_K3416102,BBa_K3416103. Primers to amplify a fragment of 16S rRNA are:

F_Col: CAGGGGGATAGCCCAGAGAAATTTGG

R_Col: ACCACACCAACTAGCTAATGGGACGC

In our case, detection and capture probes were created to be complementary to the negative strand of the gene. All protocols needed to prepare LFA tests as well as to perform HDA can be found in Vilnius-Lithuania iGEM 2020 team wiki page.

Description of 16S F. columnare capture probe

BBa_K3416102 is a capture probe that is sprayed on the nitrocellulose membrane with a dispensing system such as BioDot. This sequence must be modified. Our team added a poly-A to make sure that the probe sequence itself is available for hybridization. Also a biotin moiety (bio, IDT) on the 3’ end must be added. Biotin modification is needed so that the probe could be immobilized on the test line of the lateral flow assay test strip via biotin-streptavidin non-covalent interaction.

Table 1. | Parameters of capture probe created for nucleic acid lateral flow assay test. Tm and GC% was calculated without poly-A sequence using IDT oligo analyzer tool. (A)20 marks poly-A sequence of 20 adenines. Bio means biotin modification.

Species	Probe type	Sequence and its modification	Hybridization site	Parameters
F. columnare 16S rRNA gene(AY577821)	Capture probe	GCCTCATTTGATT-(A)20-bio	181 - 193 bp	Tm = 36.5°C GC% = 38.5% size = 33 nt

Results

Specificity experiment of F. columnare identification LFA test. Tests were evaluated using 100 nM of DNA in 100 μL of running buffer II (10X SSC, 3.5% Triton X-100, 0.25% SDS, 12.5% formamide).

Figure 2. 1 - F. columnare asymmetric HDA with F_Col and R_Col primers, 2 - F. columnare symmetric HDA with F_Col and R_Col primers after denaturation, 3 - F. columnare asymmetric PCR with F_Col and R_Col primers, 4 - F. columnare symmetric PCR with F_Col and R_Col primers after denaturation, 5 - F. columnare asymmetric PCR with F_Psy and R_Psy primers, 6 - F. psychrophilum asymmetric PCR with F_Col and R_Col primers, 7 - F. piscis asymmetric PCR with F_Col and R_Col primers, 8 - E. coli asymmetric PCR with F_Col and R_Col primers, 9 - F. columnare asymmetric PCR with F_Col and R_Col primers on the test created for F. branchiophilum identification, 10 - no DNA template. CL indicates control line, TL - test line.

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References

1. Declercq, A. M., Haesebrouck, F., Van den Broeck, W., Bossier, P. & Decostere, A. Columnaris disease in fish: a review with emphasis on bacterium-host interactions. Vet Res, 44, 27 (2013).
2. Janda, J. M. & Abbott, S. L. 16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls. Journal of Clinical Microbiology, 45, 2761–2764 (2007).
3. Liu, B. & Liu, J. Methods for preparing DNA-functionalized gold nanoparticles, a key reagent of bioanalytical chemistry. Anal. Methods, 9, 2633–2643 (2017).
4. Zhang, X., Servos, M. R. & Liu, J. Instantaneous and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA Using a pH-Assisted and Surfactant-Free Route. J. Am. Chem. Soc., 134, 7266–7269 (2012).