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Journal of Clinical Microbiology, August 2008, p. 2800-2804, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00152-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Common Strains of Escherichia coli{triangledown}

Joshua Hill,1 Shilpa Beriwal,1 Ishwad Chandra,1 Vinod K. Paul,2 Aarti Kapil,3 Tripti Singh,1 Robert M. Wadowsky,4 Vinita Singh,5 Ankur Goyal,1 Timo Jahnukainen,1,{dagger} James R. Johnson,6 Phillip I. Tarr,7 and Abhay Vats1*

Department of Pediatrics, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, Pennsylvania 15213,1 Department of Pediatrics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India,2 Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India,3 Department of Pathology, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, Pennsylvania 15213,4 Department of Molecular Diagnostics, Atharva BioSciences, New Delhi 110029, India,5 Department of Medicine, VA Medical Center, Veterans Affairs Medical Center, University of Minnesota, 1 Veterans Drive, Minneapolis, Minnesota 55417,6 Department of Pediatrics and Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 631107

Received 25 January 2008/ Returned for modification 6 March 2008/ Accepted 31 May 2008


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ABSTRACT
 
We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E. coli isolates and all 22 urine samples (out of 89 samples tested) that had E. coli. This assay is rapid, low in cost, and simple to perform.


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TEXT
 
Escherichia coli can cause infections of a variety of extraintestinal sites such as the urinary tract, meninges, and bloodstream (3, 5, 8, 10-12, 21). Diagnosis of most extraintestinal E. coli (ExPEC) infections requires bacterial culture, requiring 1 to 2 days of incubation, and subsequent confirmatory testing (6, 17). Guidance as to the presence or absence of an ExPEC infection at initial presentation could be useful for a patient, especially when attempting to reduce excessive use of antibiotics. Although conventional DNA amplification using PCR can provide fast results, it is not widely used partly because it requires considerable skill and expensive equipment. Recently, loop-mediated isothermal amplification (LAMP), a rapid technique for amplifying DNA has been reported (14, 15). It requires incubation at a constant temperature (60 to 66°C for 30 to 60 min) and eliminates the need for specialized equipment or expertise. We report here the development and characterization of a LAMP assay to detect urinary E. coli.

Three sets of primers (outer, loop, and inner) were required for the E. coli LAMP assay that were designed by analyzing a conserved region of the E. coli malB gene with Primer Explorer version 3 software (http://primerexplorer.jp/elamp3.0.0/index.html) (Fig. 1). This gene is conserved across diverse lineages of E. coli and is not shared by other gram-negative bacteria except Shigella spp., based on BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/) (18). We considered this region to be appropriate because of its conservation, as well as the rarity of Shigella as a cause of extraintestinal infections.


Figure 1
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  		FIG. 1. LAMP primers. (i) Location of the LAMP primers in the E. coli malB gene. This sequence represents positions 3204 to 3407 in the E. coli GenBank sequence (GDB J01648). The F2 and Loop F primers overlap by one base, depicted by "*" and an italic "T". Also, two of the reverse primers, Loop B and B2, are contiguous and have no bases separating them, and the juncture is marked by "*" and an arrow. (ii) Sequences for the LAMP primers. The F1c and B2c sequences are the reverse complements of the F1 and B2 regions shown in Fig. 1 and are underlined.

The LAMP reaction was conducted using methods described previously, with minor modifications (14, 15). The final LAMP reaction (total, 25 µl) contained the three primer pairs in the following concentrations: 0.2 µM outer primers, 0.8 µM loop primers, and 1.6 µM inner primers. The reaction mix also contained 2.5 µl of 10x Bst DNA polymerase reaction buffer [1x containing 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH2)SO4, 2 mM MgSO4, 0.1% Triton X-100], 1 µl of an 8-U/µl concentration of Bst DNA polymerase (New England Biolabs, Inc., MA), 2 mM MgSO4 (2 µl), 5 µl of betaine (Sigma-Aldrich, St. Louis, MO), and 5 µl of sample. We generated and quantified a plasmid containing the target E. coli sequence to determine the sensitivity of the LAMP assay. The target sequence in E. coli was amplified by PCR using the primers F3 and B3 of the LAMP assay and cloned by using a StrataClone PCR cloning kit (Stratagene, La Jolla, CA). The copy number of the solution containing the E. coli clone was determined by UV spectrophotometry (2). The analytical sensitivity of LAMP was determined by using 10-fold serial dilutions of the E. coli clone.

The LAMP reaction was performed in a heating block (Lab-Line, Iowa). For comparison, it was also performed using a conventional thermal cycler (Bio-Rad, California), and both machines performed equally well. The cost per reaction was ca. 50¢ (U.S. currency).

A positive E. coli LAMP reaction typically required incubation for 60 min at 66°C to produce a ladder pattern on agarose gel (Fig. 2i). The E. coli LAMP products from several of the bands in the gel were excised and sequenced. The amplicons matched the E. coli malB gene segment spanned by F3/B3 primers. The E. coli LAMP products were also detected without electrophoresis using ambient light after the addition of a number of DNA dyes to the reaction tube. These dyes included propidium iodide, ethidium bromide, methylene blue, acridine orange, and Sybr green. Of the various dyes, Sybr green (1:10 dilution of a 10,000x stock solution) and propidium iodide (1:10 dilution of 10-mg/ml stock solution) produced the best visual discrimination. For both the dyes, 1 µl of the diluted dye was added to 25 µl of the reaction mix to develop the color reaction (Fig. 2ii). UV transillumination of the reaction mix containing the DNA dye improved the differentiation further.


Figure 2
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  		FIG. 2. LAMP assay reactions. (i) A gel image showing LAMP products from E. coli and other bacteria. The lane numbers correspond to the following specimens. Lanes: 1, E. coli; 2, 100-bp ladder; 3, Proteus mirabilis; 4, Pseudomonas aeruginosa; 5, Enterobacter faecalis; 6, Staphylococcus aureus; 7, Streptococcus pyogenes; 8, Streptococcus pneumoniae; 9, Streptococcus viridans; 10, no target control. (ii) Visualization of LAMP products by inspection. (A) Propidium iodide without UV illumination. The color changes from a deep red-orange in the negative reaction to a light (almost clear) pink in the positive reaction. (B) Propidium iodide with UV illumination showing bright transillumination in the positive reaction. (C) Sybr green without UV illumination. The color changes from orange in the negative to green in the positive reaction. (D) Sybr green with UV illumination showing bright transillumination in the positive reaction.

The specificity of the assay was evaluated by testing eight gram-negative bacteria (Acinetobacter spp., Citrobacter freundii, Enterobacter cloacae, Haemophilus influenzae, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens) and six gram-positive bacteria (Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus viridians). No ladder pattern was seen with any of the other non-E. coli bacteria or with the negative "no-target" control (Fig. 2i). The sensitivity of E. coli LAMP was found to be approximately 10 copies per reaction when an incubation period of 60 min was used. Larger copy numbers required shorter incubation times (as low as 15 to 20 min for >1,000,000 copies).

We further tested a range of E. coli isolates by this assay. A total of 36 isolates of E. coli from unique patients (n = 23) or the Escherichia coli Reference (ECOR) Collection (n = 13) were tested. ECOR (established in 1984) contains 72 strains and five phylotypes (16). A LAMP assay was performed on an aliquot of bacterial solution without extraction of DNA. It was able to detect all of the 36 E. coli isolates (Table 1). Finally, 89 fresh urine samples were tested by LAMP assay, and the results were compared to cultured urine samples obtained from microbiology laboratories of the Children's Hospital of Pittsburgh and the All India Institute of Medical Sciences from patients suspected with urinary tract infections (UTI). The culture was considered "positive" if there was growth of individual bacteria in a concentration of >100,000 colonies/ml. Growth of multiple bacteria was considered contamination. The samples were stored at 4°C after plating of urine and were analyzed by LAMP assay within 1 to 3 days of collection. Initial LAMP testing, performed on three known E. coli-positive urine samples, showed positive results both with and without DNA extraction using a QIAmp DNA Mini-Kit (Qiagen, California). Subsequent tests were performed on unextracted urine, i.e., 5 µl of unprocessed urine was directly added to the LAMP reaction mix, followed by incubation for 60 min at 66°C. Of the 89 urine samples, 22 were positive for E. coli, all of which tested positive by the LAMP assay. In addition, 36 samples had no bacterial growth, and the LAMP assay results were negative in all of these samples. Finally, in 31 samples, organisms other than E. coli grew, including 13 samples that produced ≥1 bacterial species. In 29 of these 31 samples, the LAMP assay was negative. Two non-E. coli samples that had tested positive by the LAMP assay had grown multiple organisms and probably had E. coli as one of the contaminants (Tables 2 and 3).


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  		TABLE 1. Description of isolates of E. coli evaluated by LAMP


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  		TABLE 2. Clinical comparison of LAMP and culture results from urine samples obtained from patients with suspected UTI


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  		TABLE 3. Statistical comparison of LAMP and culture results from urine samples obtained from patients with suspected UTIa

We describe here a new, rapid, and economical assay to detect E. coli in human urine in 1 h (or less), which makes this assay amenable for point-of-care or outpatient settings. There are several advantages of the LAMP over PCR, the conventional method for DNA amplification. This assay only needs a heating block and obviates the need for a thermal cycler. Also, DNA extraction is not required for LAMP. In contrast, unprocessed urine normally requires DNA extraction for PCR, since urinary urea can inhibit PCR at concentrations of >50 mM and the normal concentration of urea in adult urine is >330 mM (1, 4, 9, 20). Hence, the LAMP assay, by eliminating the step of DNA extraction, is more "user friendly" than PCR. We focused on urine as the analyte for the present study and obtained consistent results for all of the culture-positive samples. Although we did not test other biological fluids (e.g., blood, cerebrospinal fluid, etc.) it is likely, based on our recent studies on BK virus LAMP, that E. coli LAMP would perform well with these types of specimens (2).

Another advantage of this assay is that identification of a positive reaction does not require any special processing or electrophoresis. It could be detected by looking for a color change of the reaction mix in ambient light, when a DNA-binding dye is used. This visualization can be further enhanced by UV transillumination. We also showed that a simple chemical such as propidium iodide can be used for detecting a positive reaction. To our knowledge, this is the first report of using propidium iodide to visualize LAMP products. Propidium iodide is less expensive, does not require freezing for storage, and is more easily available in developing countries, such as India, than is Sybr green. In theory, a deep color of the urine (bilirubin, myoglobin, or other chromogens) could affect the color of the final LAMP products and interfere with the assay results, but we did not see this effect in the range of samples of urine tested that had minor color variation (from clear to pale yellow).

Our assay was able to detect a large number of strains studied with a very high sensitivity. Indeed, the ability to detect 10 copies per reaction may be considered to be "too sensitive" for use as a clinical assay for UTI diagnostics. However, the assay's sensitivity can be lowered to match the conventionally accepted cutoff of >100,000 copies/ml for urine if a shorter incubation time is used (i.e., 30 to 40 min versus 60 min). It is likely that different levels of sensitivity may be required for different biological fluids. For example, it may be necessary to have a high sensitivity for blood and CSF versus a lower sensitivity for urine for the assays to be clinically meaningful. Also, an ability to detect a lower copy number may be important in partially treated infections (after empirical antibiotic doses), where the organisms are reduced in number or not culturable but the DNA is still detectable.

Although LAMP assays for various specific E. coli strains have been reported, these studies were designed to detect virulence loci or markers of specific strains, such as verotoxin (7, 19, 22). However, for detection of UTI-associated E. coli strains, a less discriminatory approach is intended. Our studies extend the role of LAMP to the rapid diagnosis of such infections, using this less-biased approach. In addition to the advantages described here, there are some limitations of the assay in its current format. This assay is suited for qualitative but not quantitative information. Possibly, semiquantitative assays can be developed where the time required to develop a positive reaction could be used as a surrogate for the starting copy number. Also, LAMP assays do not provide antimicrobial susceptibility information and since bacteria demonstrate increasing resistance to antimicrobial agents, this assay is more likely to be an adjunct rather than a replacement for culture. Finally, in contrast to culture, this assay detects only a single organism. However, LAMP assays for other common bacterial pathogens, including Staphylococcus spp., have recently been reported (13); these assays may potentially be combined with this assay to screen for common infections. Further studies involving a larger number of patients and different specimens are needed in order to evaluate the clinical utility of this assay.


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ACKNOWLEDGMENTS
 
Funding for this study was provided by an NIH award (R01 AI060602) to A.V. This study was also partially supported by a special grant from the Office of the Senior Vice-Chancellor for the Health Sciences, University of Pittsburgh, and by grant 1 UL1 RR024153 from the National Center for Research Resources (NCRR), a component of the NIH and NIH Roadmap for Medical Research. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. Information on NCRR is available at http://www.ncrr.nih.gov/. Information on Re-engineering the Clinical Research Enterprise can be obtained from http://nihroadmap.nih.gov/clinicalresearch/overview-translational.asp.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pediatrics, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213. Phone: (412) 692-5182. Fax: (412) 692-7443. E-mail: abhay.vats{at}chp.edu Back

{triangledown} Published ahead of print on 11 June 2008. Back

{dagger} Present address: Hospital for Children and Adolescents, University of Helsinki, PB 281, 00029 Helsinki, Finland. Back


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REFERENCES
 
    1
  1. Behzadbehbahani, A., P. E. Klapper, P. J. Vallely, and G. M. Cleator. 1997. Detection of BK virus in urine by polymerase chain reaction: a comparison of DNA extraction methods. J. Virol. Methods 67:161-166.[CrossRef][Medline]
    2. 2
  2. Bista, B. R., C. Ishwad, R. M. Wadowsky, P. Manna, P. S. Randhawa, G. Gupta, M. Adhikari, R. Tyagi, G. Gasper, and A. Vats. 2007. Development of a loop-mediated isothermal amplification assay for rapid detection of BK virus. J. Clin. Microbiol. 45:1581-1587.[Abstract/Free Full Text]
    3. 3
  3. Bonacorsi, S., and E. Bingen. 2005. Molecular epidemiology of Escherichia coli causing neonatal meningitis. Int. J. Med. Microbiol. 295:373-381.[CrossRef][Medline]
    4. 4
  4. Chernesky, M. A., D. Jang, J. Sellors, K. Luinstra, S. Chong, S. Castriciano, and J. B. Mahony. 1997. Urinary inhibitors of polymerase chain reaction and ligase chain reaction and testing of multiple specimens may contribute to lower assay sensitivities for diagnosing Chlamydia trachomatis-infected women. Mol. Cell Probes 11:243-249.[CrossRef][Medline]
    5. 5
  5. Clarke, S. C. 2001. Diarrhoeagenic Escherichia coli: an emerging problem? Diagn. Microbiol. Infect. Dis. 41:93-98.[CrossRef][Medline]
    6. 6
  6. Farthing, M. J. 2004. Bugs and the gut: an unstable marriage. Best Pract. Res. Clin. Gastroenterol. 18:233-239.[CrossRef][Medline]
    7. 7
  7. Hara-Kudo, Y., J. Nemoto, K. Ohtsuka, Y. Segawa, K. Takatori, T. Kojima, and M. Ikedo. 2007. Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification. J. Med. Microbiol. 56:398-406.[Abstract/Free Full Text]
    8. 8
  8. Johnson, J. R. 2003. Microbial virulence determinants and the pathogenesis of urinary tract infection. Infect. Dis. Clin. N. Am. 17:261-278.[CrossRef][Medline]
    9. 9
  9. Khan, G., H. O. Kangro, P. J. Coates, and R. B. Heath. 1991. Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J. Clin. Pathol. 44:360-365.[Abstract/Free Full Text]
    10. 10
  10. Klein, E. J., D. R. Boster, J. R. Stapp, J. G. Wells, X. Qin, C. R. Clausen, D. L. Swerdlow, C. R. Braden, and P. I. Tarr. 2006. Diarrhea etiology in a children's hospital emergency department: a prospective cohort study. Clin. Infect. Dis. 43:807-813.[CrossRef][Medline]
    11. 11
  11. Leung, A. K., W. L. Robson, and H. D. Davies. 2006. Traveler's diarrhea. Adv. Ther. 23:519-527.[CrossRef][Medline]
    12. 12
  12. Marrs, C. F., L. Zhang, and B. Foxman. 2005. Escherichia coli-mediated urinary tract infections: are there distinct uropathogenic E. coli (UPEC) pathotypes? FEMS Microbiol. Lett. 252:183-190.[CrossRef][Medline]
    13. 13
  13. Misawa, Y., A. Yoshida, R. Saito, H. Yoshida, K. Okuzumi, N. Ito, M. Okada, K. Moriya, and K. Koike. 2007. Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures. J. Infect. Chemother. 13:134-140.[CrossRef][Medline]
    14. 14
  14. Nagamine, K., T. Hase, and T. Notomi. 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell Probes 16:223-229.[CrossRef][Medline]
    15. 15
  15. Notomi, T., H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino, and T. Hase. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28:E63.[CrossRef][Medline]
    16. 16
  16. Ochman, H., and R. K. Selander. 1984. Standard reference strains of Escherichia coli from natural populations. J. Bacteriol. 157:690-693.[Abstract/Free Full Text]
    17. 17
  17. Russo, T. A., and J. R. Johnson. 2003. Medical and economic impact of extraintestinal infections due to Escherichia coli: focus on an increasingly important endemic problem. Microbes Infect. 5:449-456.[CrossRef][Medline]
    18. 18
  18. Shaikh, N., and P. I. Tarr. 2003. Escherichia coli O157:H7 Shiga toxin-encoding bacteriophages: integrations, excisions, truncations, and evolutionary implications. J. Bacteriol. 185:3596-3605.[Abstract/Free Full Text]
    19. 19
  19. Song, T., C. Toma, N. Nakasone, and M. Iwanaga. 2005. Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediated isothermal amplification method. FEMS Microbiol. Lett. 243:259-263.[CrossRef][Medline]
    20. 20
  20. Toye, B., W. Woods, M. Bobrowska, and K. Ramotar. 1998. Inhibition of PCR in genital and urine specimens submitted for Chlamydia trachomatis testing. J. Clin. Microbiol. 36:2356-2358.[Abstract/Free Full Text]
    21. 21
  21. Trotman, H., and Y. Bell. 2006. Neonatal sepsis in very low birthweight infants at the University Hospital of the West Indies. West Indian Med. J. 55:165-169.[Medline]
    22. 22
  22. Yano, A., R. Ishimaru, and R. Hujikata. 2007. Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by loop-mediated isothermal amplification. J. Microbiol. Methods 68:414-420.[CrossRef][Medline]

Journal of Clinical Microbiology, August 2008, p. 2800-2804, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00152-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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