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Journal of Clinical Microbiology, March 2004, p. 1236-1242, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1236-1242.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Hairpin Primers for Simplified Single-Nucleotide Polymorphism Analysis of Mycobacterium tuberculosis and Other Organisms

Manzour Hernando Hazbón* and David Alland

Division of Infectious Diseases, Department of Medicine, and the Ruy V. Lourenço Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103

Received 18 August 2003/ Returned for modification 12 October 2003/ Accepted 5 December 2003

We describe a novel, simple, rapid, and highly sensitive method to detect single-nucleotide polymorphisms (SNPs) in Mycobacterium tuberculosis and other organisms. Amplification refractory mutation (ARMS) SNP assays were modified by converting the SNP-detecting linear primers in the ARMS assay to hairpin-shaped primers (HPs) through the addition of a 5' tail complementary to the 3' end of the linear primer. The improved ability of these primers to detect SNPs in M. tuberculosis was compared in a real-time PCR with SYBR-I green dye. Linear primers resulted in incorrect or indeterminate allele designation for 6 of the 13 SNP alleles tested in seven different SNP assays, while HPs determined the correct SNP in all cases. We compared the cycle threshold differences ({Delta}Ct) between the reactions containing primer-template matches and the reactions containing primer-template mismatches (where a larger {Delta}Ct indicates a more robust assay). The use of HPs dramatically improved the mean {Delta}Ct values for the SNP assays (7.6 for linear primers and 11.2 for HPs). We designed 98 different HP assays for SNPs previously associated with resistance to the antibiotic isoniazid to test the large-scale utility of the HP approach. Assay design was successful in 72.4%, 83.7%, 88.8%, and 92.9% of the assays after one to four rounds of assay design, respectively. HP SNP assays are simple, sensitive, robust, and inexpensive. These advantages favor the application of this technique for SNP assays of M. tuberculosis and other organisms.

* Corresponding author. Mailing address: Division of Infectious Diseases, Department of Medicine, and the Ruy V. Lourenço Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Ave., MSB A900, Newark, NJ 07103. Phone: (973) 972-2175. Fax: (973) 972-0713. E-mail: hazbonmh{at}umdnj.edu.

Journal of Clinical Microbiology, March 2004, p. 1236-1242, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1236-1242.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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