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Research Article
Evaluation of Polymerase Chain Reaction for Direct Detection of Escherichia coli Strains in Environmental Samples

Safa A. Sherfi, Hamid A. Dirar, Badr E. Hago, Mohamed E. Ahmed, Hassan A. Musa, Hassan Abu Aisha and Imadeldin E. Aradaib

Research Journal of Microbiology, 2007, 2(2), 163-169.

Abstract

The potential of the Polymerase Chain Reaction (PCR), as a means of detecting Escherichia coli (E. coli) DNA in suspected environmental samples, was studied. Using a pair of outer primers P1 and P2, selected from uidA gene, which encodes E. coli glucuronidase, the PCR-based assay resulted in amplification of a 486 base pair (bp) PCR product. E. coli strains from different environmental sources including recycled and drinking water as well as stagnant water were detected by this nested PCR-based assay. Amplification products were visualized on ethidium bromide-stained agarose gel. The sensitivity of the PCR assay was 100 fg of bacterial DNA with ethidium bromide-stained agarose gels. Using a pair of internal (nested) primers P3 and P4, the nested PCR produced a 186 bp PCR product. The nested PCR increased the sensitivity of the PCR assay by 1,000 times and specific PCR products were detected from 0.1 fg of bacterial DNA. Amplification product was not detected when the nested PCR-based assay was applied to DNA from other related bacteria including, Salmonella, Pseudomonas and Proteus or nucleic acid-free water. Application of this nested PCR-based assay to environmental samples resulted in direct detection of E. coli DNA from sewage water, tap water, drinking water at Shambat Campus, University of Khartoum, Sudan. This nested PCR-based assay should provide a rapid, sensitive and specific assay for direct detection and quantification of E. coli in environmental samples suspected to contain the organism.

ASCI-ID: 83-109

Fig. 1). Using the internal primers (P3 and P4), the nested PCR resulted in amplification of a 186 bp PCR product. The nested 186 bp PCR product was detected from as little as 0.1 fg of E. coli DNA target (Fig. 2). Using 1 pg of E. coli DNA target extracted from purified colonies, the primary 486 bp specific PCR products was detected in eighteen E. coli strains isolated from different environmental sources (Fig. 3).

Fig. 1: Sensitivity of the polymerase chain reaction for detection of the primary 486-bp PCR product from different DNA concentrations of E. coli. Visualization of the 486-bp specific- PCR product on ethidium bromide-stained agarose gel from 100 fg DNA. Lane MW: molecular weight marker (100 bp ladder); lanes 1-7: E. coli DNA 100, 10 and 1.0 pg, 100, 10, 1.0 and 0.1 fg, respectively

Fig. 2: Sensitivity of the nested polymerase chain reaction (nPCR) for detection of the nested 186 bp PCR product from E. coli. Visualization of the 186-bp specific-PCR product on ethidium bromide-stained agarose gel from 100 fg DNA. Lane MW: molecular weight marker; lanes 1-7: E. coli DNA 100, 10 and 1.0 pg, 100, 10, 1.0 and 0.1 fg, respectively

Fig. 3: Visualization of the 486 bp specific E. coli PCR product on ethidium bromide-stained agarose gel from 1.0 pg of DNA of nineteen different E.coli strains. Lane MW: molecular weight marker; Lane 1-18: E. coli DNA; Lane 19: Negative control

The amount of 1.0 pg bacterial DNA extracted from closely related members of enterobacteriacae including Salmonella sp., Proteus sp. and Pseudomonas; or nucleic acid-free water failed to demonstrate the primary or the nested PCR products (Fig. 4).


Fig. 4: Specificity of the polymerase chain reaction (PCR) for E. coli DNA. The specific 486 bp PCR product was not detected from 1.0 pg of DNA from Salmonella, Proteus and Pseudomonas or nucleic acid-free water. Lane MW: molecular weight marker; Lane 1: E.coli DNA (positive control); Lane 2-4: DNA extracts from Salmonella, Proteus and Pseudomonas sp., respectively; Lane 5: nucleic acid-free water

Fig. 5: Visualization of the 186-bp specific- PCR product on ethidium bromide-stained agarose gel directly from environmental samples suspected to contain E. coli. Lane 1: Tap water; Lane 2: sewage water; Lane 3 and 4: drinking water in animal houses; Lane 5: human drinking water source; Lane 6: stagnant water; Lane 7: E. coli DNA positive control

With nested PCR amplification, the specific PCR products were detected directly from environmental water sources suspected to be contaminated with E. coli including, sewage water, tap water, drinking water collected from animal houses, stagnant water (Fig. 5). All bacterial isolates, which were PCR positive, were also found culture positive as confirmed by conventional isolation methods.

DISCUSSION

The described nested PCR assay reproducibly and specifically detected E. coli DNA directly in contaminated environmental sample and from purified colonies of the organism and directly from environmental samples. The specific 486 bp PCR products or the nested 186 bp PCR products, visualized on ethidium bromide-stained agarose gel, were obtained from all E. coli strains. The nested PCR assay was a simple procedure that efficiently detected all E. coli strains under the stringency condition used in this study. It was easier when compared to other molecular biological techniques, many of which are lengthy and cumbersome (Aradaib et al., 2005; Darby et al., 1997; Esteban and Molleja, 1998; Garner et al., 1988; Juck et al., 1996).

The sensitivity studies indicated that the nested PCR protocol was capable of detecting the amount of 0.1 fg of total E. coli genomic DNA, which corresponds to a single organism. The nested PCR assay is almost 1,000 times more sensitive than the hybridization assays (Rath and Mach, 2000).

The specificity studies indicated that, the primary specific 486 bp PCR product was not amplified from 1.0 pg of DNA extracted from different related bacteria including Salmonella, Proteus and Pseudomonas sp., or nucleic acid free water under the same stringency condition described in this study. Temperature and time for denaturation, primer annealing and extension, enzyme and magnesium chloride concentration and number of cycles of the three temperatures per time segments were very important for maintaining sensitivity and specificity of the PCR reaction. Forty cycles of amplification were used as standard PCR procedure. The amplification procedure usually takes 2 h for the primary amplification and also 2 h for nested PCR amplification. Thus, the time required for the PCR amplification was approximately 4 h.

Excellent correlation of results from the primary and nested amplification was obtained using this PCR protocol. This finding suggests that tentative diagnosis of E. coli could be based on visualization of the primary amplified 486 bp PCR product on an ethidium bromide-stained agarose gel, since it is a simple procedure that requires only 1 h after amplification. Nested amplification is necessary to confirm the specificity of the primary amplified product and to increase the sensitivity of the PCR assay particularly, when the concentration of the bacterial DNA in the suspected environmental sample is less than 100 fg. Successful amplification was also obtained directly from environmental samples with out primary isolation in culture media as shown in Fig. 5. Lane 7 of Fig. 5, which represents E. coli DNA (positive control), contains 2 bands. The 2 bands correspond to the primary 486 bp and the nested 186 bp PCR products. This could be due to high concentration of bacterial DNA in the positive control sample, which allowed selective amplification for both primary and nested PCR products. The result of this study indicated that the described nested PCR protocol has the potential to detect E. coli in environmental samples at a very low concentration. DNA extraction was a simple procedure that takes only 15 min using the commercially available QIAamp extraction Kit. To our knowledge, there is no information available on infectivity dosage. It probably requires 5 organisms to produce the nested 186 bp PCR product by the described PCR assay. This number of bacterial organisms may represent less than one infectious unit of E. coli and that could be the reason for PCR positive but bacterial culture isolation negative results. In addition, PCR detects intact organisms, injured bacteria as well as bacterial nucleic acids. Therefore, it is not uncommon to obtain PCR-positive, but bacterial isolation negative, results from the same environmental sample (Aradaib et al., 2005; Darby et al., 1997).

The E. coli nested PCR assay described in this study can replace the need for the lengthy cumbersome bacterial isolation procedures. The time required from sample submission to interpretation of the final results was consistently 8 consecutive hours, which is affordable with in the same working day. The rapidity, sensitivity and specificity of the nested PCR assay would greatly facilitate detection of E. coli in the suspected environmental samples particularly, those associated with fecal contaminations (Edberg et al., 2000; Font et al., 1997; Fricker and Fricker, 1996). Negative and positive controls should be included in each PCR reaction to estimate the lower limit of specificity and the higher limit of sensitivity. In conclusion, the described nested PCR could be used for direct detection of E. coli strains in different environments suspected of containing the organism.

ACKNOWLEDGMENTS

This study was supported by funds from the Ministry of Science and Technology, Republic of the Sudan. We are also very grateful to Mrs. Sakeena Musa, Mrs. Lana Mustafa and Mr. Adalla M. Fadlelmula for technical assistance.

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