Single primer designed for detection of Shigella species

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1 African Journal of Microbiology esearch Vol. 6(5), pp , 5 July, Available online at DOI:.5897/AJM.33 ISSN Academic Journals ull Length esearch Paper Single primer designed for detection of Shigella species athina Kumar Shanmugakani and Asit. Ghosh * M.Tech.Biotechnology, School of BioSciences and Technology, VIT University, Vellore-63 4, Tamilnadu, India. School of BioSciences and Technology and Centre for Infectious Diseases and Control, VIT University, Vellore 63 4, Tamilnadu, India. Accepted 5 April, In infectious disease diagnosis prior to treatment, identification of the causative agent plays a crucial role. Polymerase chain reaction (PC) has paved a molecular way for specific species identification. In this study, we have attempted to design a single common primer to identify the causative agent for shigellosis - Shigella spp. Bioinformatic tools like CLUSTALX for multiple sequence alignment, Primer3 for primer designing, NetPrimer for primer analysis, in silico PC amplification tool for amplification were used. In silico PC amplification gave the desired amplicon with the same sequence that had been used for primer designing. A single primer was thus designed for the genus Shigella and is predicted to be a prototype sequence for easy and rapid detection of Shigella spp. Key words: Virulence factor data base (VDB), CLUSTALX, Primer3, NetPrimer, in silico polymerase chain reaction (PC) amplification. INTODUCTION Shigellosis, the bacillary dysentery is caused by Shigella spp. Shigella dysentriae, Shigella flexneri, Shigella sonnei and Shigella boydii are the four species of Shigella (Castellani and Chalmers, 99). S. flexneri and S. dysenteriae are predominant in the tropical developing countries and S. sonnei prevalent in the industrialized countries, are majorly involved in affecting massive number of people (Ghosh and Sehgal, 998). Every year 64.7 million people are afflicted by shigellosis in both developing and developed countries with. million deaths only from developing countries (Kotloff et al., 999). Besides the fatality posed by shigellosis, it possess pandemic potentials with ever-increasing multiple drug resistance (Ghosh et al., 3). The infectious dose of Shigella spp. is very low ( to cells) as when compared with other enteric pathogens like Salmonella, Escherichia coli, Vibrio cholerae O or/and O39 ( 5 to 9 cells) (Crockett et al., 996). *Corresponding author. asitranjanghosh@vit.ac.in, asitranjan@gmail.com. Tel: , Hence shigellosis is a prime concern in public health. The identification of Shigella spp. from fecal samples by conventional microbiological analysis majorly offers phenotypic characterization. Genotypic characterization supersedes the scientific gain over phenotypic analyses. Tools for genotypic identification have brought a numerable success over phenotypic characterization like clonality determination, trafficking infections, etc (oy et al., 5). But identification of Shigellae is still based on the conventional microbiological methods using the battery of culture media, biochemical tests, etc. which requires many media, chemicals, reagents, expertise and time. Polymerase chain reaction (PC) overcomes the difficulties in phenotypic characterization. It results in the amplification of a specific nucleotide sequence of interest, so that the organism can be identified genetically (Lampel et al., 99). A primer had been designed in such a way that it can amplify a region of the genomic DNA in all four Shigella spp. or identification of more than one species, it is necessary to identify a long run of conserved sequences in all the test organisms (Grimont et al., 7). Hence a single primer can amplify a common sequence in the

2 Shanmugakani and Ghosh 589 igure. Similar sequences in seven Shigella species. genome of all organisms. or effective amplification, the primer for amplification reaction should be optimal with desired characteristics like GC content, melting temperature and without secondary structures. The primers were designed with the desired characteristics and checked for the formation of secondary structures like hairpin loops and self dimer. It upgraded the validation of a number of primers that best suited the template and gave optimal output (Maheux et al., 9). MATEIALS AND METHODS Virulence factor data base (VDB) contains the whole genomic and plasmid DNA sequences of pathogens (Chen et al., 5). Genomic DNA sequences of seven pathogenic Shigella strains viz. S. dysentriae (NC_7), S. sonnei (NC_7384), serotypes of S. boydii (NC_658 and NC_763) and 3 serotypes of S. flexneri (NC_474, NC_4337 and NC_858) were retrieved from VDB. The retrieved sequences were run into CLUSTALX. It is a multiple sequence alignment tool for long sequences. Once the nucleotide sequences were run into this tool, it showed the similar nucleotides in all the given input sequences by the symbol *(asterix). or different parameters GC content, melting temperature, length of the primers set in Primer3 tool, 5 sets of forward and reverse primers were designed for those similar nucleotide sequences. Net primer was used to check the formation of hairpin loops and self dimers by the designed primers. Then the primer that is devoid of hairpin loops and self dimer was chosen and checked for in silico amplification using the in silico PC amplification tool. ESULTS CLUSTALX showed a continuous length of 36 bases similar in all the seven input sequences as shown in igure. 5 forward and 5 reverse primers were designed by Primer3 tool for these 36 bases as shown in Table. On analyzing these 5 primers with NetPrimer, only one set of forward and reverse primer were not forming hairpin loops and self dimer as shown in Table. It was selected and designated as shrkag which is shown as follows: orward primer: 5 -AACTGGTTACCTGCCGTGAG-3 everse primer: The amplification by the primer shrkag in all the shigella species using the in silico PC amplification tool is shown in igure. The result showed that there is no amplification in S. sonnei. It was shown that the amplicon is the same similar sequences in all the Shigella species that had been taken for primer designing. Hence there is more possibility that this primer will work in laboratory approach also. DISCUSSION The primer thus designed can be used for identification of

3 59 Afr. J. Microbiol. es. Table. Output of designed sets of primers for the detection of Shigella spp.using Primer3. Start point Primer type Primer sequences 5-3 Size Product size T m ( C) GC (%) , orward primer;, reverse primer. G TGCAACGGGCAATATGTC T CTGCAACGGGCAATATGTC GCGTTTCATGGATGTTGT GGTGCTAATGCGTTTCAT CGGGCAATATGTCTCTGT ACGGGCAATATGTCTCTG GCAATATGTCTCTGTGTGGA GCAATATGTCTCTGTGTGGAT TTCTGTGTTTCCTGTACGC AGCAGCTTCTGAACTGGTTA CT CT TGGTGGTAATGGTGATGGTG AACTGGTTACCTGCCGTGAG GGCAATATGTCTCTGTGTGG CTAATGCGTTTCATGGATGT ACTGGTTACCTGCCGTGAGT TGGTGCTAATGCGTTTCA TGCTAATGCGTTTCATGG TGCGTTTCATGGATGTTG CTGCAACGGGCAATATG

4 Shanmugakani and Ghosh 59 Table. Output of designed sets of primers for secondary structures using NetPrimer. Start point Primer type Primer sequences 5-3 Hairpin loops Self dimer , orward primer;, everse primer. G TGCAACGGGCAATATGTC T CTGCAACGGGCAATATGTC GCGTTTCATGGATGTTGT GGTGCTAATGCGTTTCAT CGGGCAATATGTCTCTGT ACGGGCAATATGTCTCTG GCAATATGTCTCTGTGTGGA GCAATATGTCTCTGTGTGGAT TTCTGTGTTTCCTGTACGC AGCAGCTTCTGAACTGGTTA CT CT TGGTGGTAATGGTGATGGTG AACTGGTTACCTGCCGTGAG GGCAATATGTCTCTGTGTGG CTAATGCGTTTCATGGATGT ACTGGTTACCTGCCGTGAGT TGGTGCTAATGCGTTTCA TGCTAATGCGTTTCATGG TGCGTTTCATGGATGTTG CTGCAACGGGCAATATG

5 59 Afr. J. Microbiol. es No bands () () () () () () igure. In silico amplification by shrkag., Shigella flexneri a str 3;, Shigella flexneri a str. 457T; 3, Shigella sonnei Ss4; 4, Shigella flexneri 5 str. 84; 5, Shigella dysenteriae Sd97; 6, Shigella boydii CDC the seven Shigella spp. Here the nucleotides in the genomic DNA sequence is taken under consideration. But if the plasmid DNA sequences are used, there is a possibility of low yield because the viability of the plasmids is not well defined. Also the plasmid profile analysis showed the similarity between Shigella spp. Hence a single primer specific for the particular genus is required. Since the pathogenicity of both the genus Escherichia coli and Shigella will lead to dysentry, when the fecal samples are analysed, there may be ambiguities in the discrimination of E. coli from Shigella. The amplification with the same primer to E. coli showed positive results except for two strains. Hence it showed the similarity between E. coli and Shigella. In other words, if the microbe had been identified in the first step itself then the further processing of treatment will become very easy. Otherwise, we have to look for the biochemical characteristics of the microbes which may take time and also not with % specificity. It was cross examined for amplification in other microbes like Klebsiella, Yersinia, Salmonella, Staphylococcus, Lactobacillus, Enterobacter, Enterococcus etc.. All of them showed no bands of amplification in in silico PC amplification. Since PC is mainly concerned with the nucleotide sequences rather than the function they codes for, this primer can be used for the isolation of multigeneric microbes in a single reaction. or example, if an environmental sample is to be analysed we have to go for plating, biochemical characterization etc.. But if we subject that sample to PC in the first step itself with the designed primer, limited number of species can be scrutinized. rom that we can characterize the specific microbe from the crude sample. The subjection of the samples to biochemical

6 Shanmugakani and Ghosh 593 characterization will be a time consuming process. In clinical laboratories, people are looking for the rapid detection of the pathogen in the given sample. Hence PC can be an effective process for species identification in terms of both rapidity and specificity. EEENCES Castellani A, Chalmers AJ (99). Manual of tropical medicine. Williams Wood & Co., New York, 3rd ed. pp Chen L, Yang J, Jun Yu, Yao Z, Sun L, Shen Y, Jin Q (5). VDB: a reference database for bacterial virulence factors. Nucleic Acids es., 33: D35 D38. Crockett CS, Haas CN, azil A, ose JB, Gerba CP (996). Prevalence of shigellosis in the U.S.: consistency with dose-response information. Int. J. ood Microbiol., 3(-): Ghosh A, Sehgal SC (998). Shigella infections among children in Andaman - an archipelago of tropical islands in Bay of Bengal. Epidemiol. Infect., : Ghosh A, Sugunan AP, Sehgal SC (3). Emergence of Nalidixic Acid-esistant Shigella sonnei in Acute-Diarrhea Patients on Andaman and Nicobar Islands, India. Antimicrob. Agents Chemother., 47(4): 483. Grimont, Collin ML, Talukder KA, Carle I, Issenhuth S, oux KL, Grimont PA (7). Identification of a group of shigella-like isolates as Shigella boydii. J. Med. Microbiol., 56(pt 6): Kotloff KL, Winickoff JP, Ivanoff B, Clemens JD, Swerdlow DL, Sansonetti PJ, Adak GK, Levine MM (999). Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull. WHO., 77(8): Lampel KA, Jagow JA, Trucksess M, Hill WE (99). Polymerase Chain eaction for Detection of Invasive Shigella flexneri in ood. Appl. Environ. Microbiol., 56(6): Maheux A, Picard J, Boissinot M, Bissonnette L, Paradis S, Bergeron MG (9). Analytical Comparison of nine PC primer sets designed to detect the presence of Escherichia coli/shigella in water samples. Water es., 43(): oy S, Dutta B, Ghosh A, Sugunan AP, Nandy K, Bhattacharya SK, Sehgal SC (5). Molecular tracking of the lineage of strains of Vibrio cholerae O biotype El Tor associated with a cholera outbreak in Andaman and Nicobar Islands,India. Trop. Med. Int. Health, (6): 64-6.