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 Table of Contents  
RESEARCH LETTER
Year : 2014  |  Volume : 6  |  Issue : 5  |  Page : 231-233

DNA extraction from nocardia species for special genes analysis using PCR


1 Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Bacteriology and Virology, Alborz University of Medical Sciences, Karaj, Iran

Date of Web Publication21-May-2014

Correspondence Address:
Seyyed Saeed Eshraghi
Department of Microbiology, Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1947-2714.132943

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  Abstract 

Background : Nocardia species have a complex cell wall structure similar to that of mycobacteria, and the extraction of DNA from this
bacterium is extremely diffi cult. Currently, to identify Nocardia species particularly, it is essential to utilize molecular techniques. Aims : In
the present study, we investigated STET (sodium chloride-TRIS-EDTA-triton) buffer for the extraction of high-quality genomic DNA from 20 clinical and environmental isolates. Materials and Methods: The extracted DNA was evaluated for portion of the 16S rRNA, 65-kDa heat-shock protein and 16S rRNA genes via polymerase chain reaction. Results: The extracted DNA had high molecular mass, and its concentration and purity was suitable when tested in 1% agarose gel, and using UV spectrophotometry. Amplifi cation of three different genes was successfully performed. Conclusion: This paper reveals an inexpensive, reproducible and effi cient method of DNA extraction from Nocardia species, which is appropriate for accurate identifi cation of this bacterium via polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism.

Keywords: 16S rRNA, DNA extraction, hsp65, Nocardia, PCR


How to cite this article:
Bafghi MF, Eshraghi SS, Heidarieh P, Habibnia S, Nasab MR. DNA extraction from nocardia species for special genes analysis using PCR. North Am J Med Sci 2014;6:231-3

How to cite this URL:
Bafghi MF, Eshraghi SS, Heidarieh P, Habibnia S, Nasab MR. DNA extraction from nocardia species for special genes analysis using PCR. North Am J Med Sci [serial online] 2014 [cited 2021 Apr 22];6:231-3. Available from: https://www.najms.org/text.asp?2014/6/5/231/132943


  Introduction Top


Nocardia species are gram-positive, partially acid-fast and non-motile bacteria that often form branched hyphae in both tissues and culture. Nocardia can be found around the world as saprophytic component of the normal soil microflora, marine water, dust, and air. Nocardial infections occur in both immunocompetent and immunosuppressive individuals, and respiratory tract is the primary site of nocardiosis. [1],[2],[3],[4] In recent years, polymerase chain reaction (PCR) sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) are used for the accurate identification of Nocardia species. [3],[4],[5],[6] Various methods, such as commercial kits and enzymatic methods (lysozyme, proteinase K), have been introduced for genomic DNA extraction from Nocardia. Enzymatic lysis is very expensive and time-consuming, thus molecular laboratories are searching for simple, inexpensive, rapid and acceptable methods for DNA extraction from bacteria. [7] Different methods have been introduced for DNA extraction from prokaryotic cells. [8],[9] In this study, we investigated STET (sodium chloride-TRIS-EDTA-triton) buffer for DNA extraction from Nocardia. [10]


  Materials and Methods Top


In this report, we describe a simple protocol for DNA extraction from the genus Nocardia using STET buffer. Pure colonies were picked from nutrient agar plate and inoculated in 5 mL of tryptic soy broth (TSB). The tube was incubated at 37°C and shaken until the turbidity of the bacterial suspension was adjusted to match 1.0 McFarland standard (approximately 3 × 10 8 bacterial cells). Bacterial suspension was pelleted via centrifugation at 13000 rpm for 5 min. The pellet was washed with sterile distilled water and re-suspended in 200 μL of STET buffer (10 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, 5% [v/v] Triton X100, pH 8.0), and the cell suspension was vortexed vigorously. The cell suspension was boiled at 100°C for 30 min and then centrifuged at 10000 rpm for 10 min. Supernatant fluid was transferred into a sterile Eppendorf tube. Subsequently, cold 95% ethanol was added to the supernatant and kept at −20°C for 60 min. After this stage, the solution was centrifuged at 13000 rpm for 10 min, the supernatant fluid was discarded, and DNA pellets were dried. DNA template was dissolved in 50 μL sterile distilled water and stored at −20°C until the PCR amplification. Purity and quality of the nucleic acid were determined by measuring A260/A280 ratio using UV spectrophotometry, and electrophoresis in 1% agarose gel (10 μL of DNA sample). PCR amplification of DNA was performed using partial sequence of 16S rRNA gene, 65-kDa heat-shock protein (hsp65) gene and 16S rRNA gene (universal bacterial 16S rRNA gene) [Table 1]. Following PCR amplification, 5 μL of PCR products were electrophoresed in 1.5% agarose gel at 70 V for 100 min in TBE buffer (Tris-HCl, Boric acid, EDTA), and stained with ethidium bromide (EtBr).
Table 1: Primers used for PCR amplifi cation in our study

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  Results and Discussion Top


Chromosomal DNA was extracted from 20 clinical and environmental isolates within 110 minutes. Using this method, the extracted DNA gave an A260:A280 ratio of 1.9:2.0, and the concentration of DNA was 142.35 μg/mL. The extracted DNA had a high molecular weight in 1% agarose gel electrophoresis [Figure 1]. PCR amplification of hsp65, partial sequence of 16S rRNA and 16S rRNA gene (universal primer) regions yielded 439-bp, 999-bp,and 1500-bp fragments, respectively (data not shown). Details are shown in [Figure 2]. The rapid method described here could be used as an effective method for DNA extraction from Nocardia species. Other procedures for DNA extraction from the genus Nocardia are expensive and time-consuming. [12],[13],[14] Loeffelholz and Scholl in 1989 established a difficult method for DNA extraction from Nocardia species. This procedure was time-consuming and monotonous. [5] Recently, STET buffer solution has been used for other bacteria, including Lactobacillus species, Clostridium perfringens, and Listeria monocytogenes. [15],[16],[17] In the present paper, we report a method for DNA extraction from Nocardia species that has not been used before for this specific bacteria. This DNA extraction method using STET buffer showed acceptable and satisfactory results for molecular epidemiology techniques such as PCR and PCR-RFLP. The described method is simple, fast, cost-effective, sensitive, and highly reproducible for DNA extraction from Nocardia, and there is no need for a skillful specialist to perform this method.
Figure 1: 10 μl of DNA template electrophoresed in 1% agarose gel

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Figure 2: Lane 1, 50-bp DNA ladder; PCR amplifi cation of the 999- bp (lane 2) and 439-bp (lane 3) fragments

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  Acknowledgments Top


This study was supported by Tehran University of Medical Sciences, Deputy of Research.

 
  References Top

1.Bafghi MF, Eshraghi SS, Heidarieh P, Habibnia S, Nasab MR. Nocardiosis in immune disorder disease. Malays J Med Sci 2014;21:75-6.  Back to cited text no. 1
    
2.Eshraghi SS. Molecular typing of Nocardia species. J Med Bacteriol 2012;1:38-45.  Back to cited text no. 2
    
3.Brown-Elliott BA, Brown JM, Conville PS, Wallace RJ Jr. Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy. Clin Microbiol Rev 2006;19:259-82.   Back to cited text no. 3
    
4.Shojaei H, Hashemi A, Heidarieh P, Eshraghi S, Khosravi AR, Daei Naser A. Clinical isolation of Nocardia cyriacigeorgica from patients with various clinical manifestations, the first report from Iran. Med Mycol J 2011;52:39-43.   Back to cited text no. 4
    
5.Loeffelholz M, Scholl D. Method for improved extraction of DNA from Nocardia asteroides. J Clin Microbiol 1989;27:1880-1.  Back to cited text no. 5
    
6.Valenzuela-Tovar JF, Contreras-Pérez C, Shibayama-Hernández H, Chávez-González L, Vázquez-Chacón CA, Olivera-Díaz H. Biochemical identification and molecular characterization of Nocardia isolates from sputum. Arch Med Res 2005;36:356-61.  Back to cited text no. 6
    
7.Conville PS, Fischer SH, Cartwright CP, Witebsky FG. Identification of Nocardia species by restriction endonuclease analysis of an amplified portion of the 16S rRNA gene. J Clin Microbiol 2000;38:158-64.  Back to cited text no. 7
    
8.Salgado A, Ramírezb N, Sandovala E, Sandoval H. Fast method for DNA extraction in Nocardia and Saccharomonospora. J Mycol Med 2008;18:100-102.  Back to cited text no. 8
    
9.Pitcher D, Saunders NA, Owen RJ. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 2008;8:151-6.  Back to cited text no. 9
    
10.Millar BC, Jiru X, Moore JE, Earle JA. A simple and sensitive method to extract bacterial, yeast and fungal DNA from blood culture material. J Microbiol Methods 2000;42:139-47.  Back to cited text no. 10
    
11.Rodríguez-Nava V, Couble A, Devulder G, Flandrois JP, Boiron P, Laurent F. Use of PCR-restriction enzyme pattern analysis and sequencing database for hsp65 gene-based identification of Nocardia species. J Clin Microbiol 2006;44:536-6.  Back to cited text no. 11
    
12.Bollet C, Gevaudan MJ, de Lamballerie X, Zandotti C, de Micco P. A simple method for the isolation of chromosomal DNA from gram positive or acid-fast bacteria. Nucleic Acids Res 1991;19:1955.  Back to cited text no. 12
    
13.Torres RD, Oletta CA, Zlotnik H. A rapid and gentle method for isolation of genomic DNA from pathogenic Nocardia spp. Clin Diagn Lab Immunol 1996;3:601-4.  Back to cited text no. 13
    
14.Chun J, Goodfellow M. A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol 1995;45:240-5.  Back to cited text no. 14
    
15.Gevers D, Huys G, Swings J. Applicability of rep PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiol Lett 2006;205:31-6.  Back to cited text no. 15
    
16.Amagliani G, Omiccioli E, Campo A, Bruce IJ, Brandi G, Magnani M. Direct detection of Listeria monocytogenes from milk by magnetic based DNA isolation and PCR. J Appl Microbiol 2004;21:597-603.   Back to cited text no. 16
    
17.Ahsani M, Mohammadabadi MR, Shamsaddini MB. Clostridium perfringens isolate typing by multiplex PCR. JVATTD 2010;16:573-8.  Back to cited text no. 17
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1]


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