[논문]A New Multiplex-PCR for Urinary Tract Pathogen Detection Using Primer Design Based on an Evolutionary Computation Method

Garcia, Liliana Torcoroma; Cristancho, Laura Maritza; Vera, Erika Patricia; Begambre, Oscar

doi:10.4014/jmb.1406.06079

A New Multiplex-PCR for Urinary Tract Pathogen Detection Using Primer Design Based on an Evolutionary Computation Method 원문보기

Journal of microbiology and biotechnology, v.25 no.10, 2015년, pp.1714 - 1727

Garcia, Liliana Torcoroma (Program of Bacteriology and Clinical Laboratory, Universidad de Santander - UDES) , Cristancho, Laura Maritza (Program of Bacteriology and Clinical Laboratory, Universidad de Santander - UDES) , Vera, Erika Patricia (Program of Bacteriology and Clinical Laboratory, Universidad de Santander - UDES) , Begambre, Oscar (School of Civil Engineering, Universidad Industrial de Santander)

Abstract ▼ AI-Helper

This work describes a new strategy for optimal design of Multiplex-PCR primer sequences. The process is based on the Particle Swarm Optimization-Simplex algorithm (Mult-PSOS). Diverging from previous solutions centered on heuristic tools, the Mult-PSOS is selfconfigured because it does not require the definition of the algorithm's initial search parameters. The successful performance of this method was validated in vitro using Multiplex-PCR assays. For this validation, seven gene sequences of the most prevalent bacteria implicated in urinary tract infections were taken as DNA targets. The in vitro tests confirmed the good performance of the Mult-PSOS, with respect to infectious disease diagnosis, in the rapid and efficient selection of the optimal oligonucleotide sequences for Multiplex-PCRs. The predicted sequences allowed the adequate amplification of all amplicons in a single step (with the correct amount of DNA template and primers), reducing significantly the need for trial and error experiments. In addition, owing to its independence from the initial selection of the heuristic constants, the Mult-PSOS can be employed by non-expert users in computational techniques or in primer design problems.

주제어

AI 본문요약
AI-Helper

제안 방법

[9] and [28] with some modifications. One of the main differences between these works and the current study is that the optimization technique employed in this study does not require the definition of any heuristic parameter (mutation probability, crossover rate, and selection rules in genetic algorithms, or cognitive parameters and inertia weight in PSO) in order to obtain a solution.

이론/모형

This primer pair, as well as its restrictions, is used for the second optimization process. Both of the two optimization processes are carried out using an adapted PSOS algorithm [7], called Mult-PSOS. For the sake of precision, each step is explained in detail in the next section.
DNA sequences from genes of uropathogenic bacteria consigned in the GenBank (http://www.ncbi.nlm.nih.gov/genbank) database were used as templates for in silico primer design experiments by PSOS and for in vitro PCR assays (Table 1). The selection of the bacteria and the genes was made according to Ref.
In this study, a set of Multiplex-PCR primers for uropathogenic bacteria detection were designed using a Mult-PSOS tool. In Tables 2 and 3, the sequences and some of the theoretical primer design parameters calculated by Gene Runner 3.
1 TOPO (Invitrogen Life Technologies). The sequence of the insert was confirmed by DNA sequencing and a search by homology was then performed using the BLASTn algorithm (http://www.ncbi.nlm.nih.gov/BLAST) [4]. For the verification of the primer specificity, extracted DNA from Serratia marcescens, Salmonella spp.
(b)). To overcome this drawback, we adopted in this work the concept of a self-configured algorithm [7]. This form of algorithm combines two heuristic methods.
The results obtained with the proposed algorithm were compared with the primers described by Ref. [28] and the oligonucleotides obtained using the Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).

성능/효과

In accordance with the results, we can conclude that Mult-PSOS is an autoconfigured algorithm that works in a sequential manner, facilitating the efficient prediction and selection of the primer sequences for the simultaneous replication of up to seven genes
Using the restriction parameters, 14 oligonucleotide sequences were selected by the algorithm as the optimal sense and anti-sense primers. In the in vitro Multiplex-PCR, amplification of all the target sequences was achieved in only two tests, validating the good performance of the Mult-PSOS. It is worth mentioning that in the first assay, five of the six amplicons (K.
It is worth highlighting that the AG results are dependent on these values. In this work, we implemented an adjusted hybrid algorithm of Particle Swarm Optimization-Simplex [7], the Mult-PSOS, in order to obtain an autoconfigured computational strategy, which allows the rapid and reliable prediction of a set of oligonucleotides for simultaneous amplification of multiple genes in a single PCR (Multiplex-PCR). This strategy consists of the selection of a first primer pair (and subsequent pairs), following all the restrictions required for an adequate primer design.
The optimization of the reaction conditions for the adequate amplification of all amplicons was accomplished in a single step (with the correct amount of DNA template and primers), decreasing significantly the need for the multiple testing by trial and error
In this way, the most penalized restriction violations were the appearance of the primer sequence more than once in a target DNA (Uni(Pt)), and the size difference among amplicons (ladM(Pt)). The second highest penalizing value was given to the factors that affect the sensitivity of the oligonucleotides, such as hairpin loop structures (self-complementary sequences) and Ta and Tm differences greater than 5℃ among primers.
coli genetic material obtained by the boiling-DNA extraction method. With this modification, 100% of the amplicons were amplified.

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표제어: PCR

동의어: Packet Collision Rate

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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