In dian Journal Expe rimenta l Biol ogy Vo l. 4 1, Fe brua ry 2003, pp. 177 - ISO Detection of spores of Bacillus anthracis from environment us in g polymerase chain reaction Syed Imt eyaz Alam l , Gauri Shanka r Agarwa l 2 , Dev Vrat Kambo/, Ga nga Prasad Rai 2 & Lokendra Singh l * IDi vision of Bio tce hn ology and 2 Di vis ion of Microb iology, Defencc R & Es tablishment. Jh ansi Ro ad. Gwa li or 474 002. In dia Recei ved 7 J ll il e 2002: revised 22 November 2002 A se nsiti ve PC R ha sed detection of" I Ja ci llus all/ limcis spores fro m e nv ironmc nt was standard izcd. Spcc ifi c 1247bp a mpli con could be dctcctcd with tc mp latc conccntrat ion as low as 13 pg. Sens iti vi ty was enhanced to 10 fold by ncsting wit h second sct of pri me rs, formin g 20Sbp a mp li con. Extraction of DN A from spores purified fr om soil sa mp les by aqucous polymcr two- ph ase system fo ll owed by pa rt ia l ge rmi nati on a nd freeze-th aw treat me nt yie ld ed bcst rcsults. Soil sam ple spiked wit h sporcs (S x 102/g of samplc) could be dctected with thi s mcth od . Bacillus anth racis is a Gram-positive spore - fo rming bacillus that ca n cause acute in fec ti on in both animals and humans l . It is prim aril y a di sease of herbivores, which acquires infec ti on a ft er co ming into co ntact with so il borne spores. Th e distribution of anthrax is worldwide, with foci of anthrax existing in the United St ates in areas of Kansas and Okl ahoma. Loc i of an- thrax also exist in the southe rn penins ul ar India with of recurring outbreaks 2-3 . Th e di sease ca n be trans mi tted to hum ans when spores of B. anthracis are introduced by in ge s ti on, inhala ti on or co ntact with the skin 4. Cutaneo us a nthr ax is the mos t co mmon fo rm of human disease and may occ ur either in industrial or ag ri cultural se ttings after expos ur e to co ntaminated animals or their products. Vir ulent stra in s of B. a l1- th ra cis are encaps ul ated and ca use d ea th in hum a ns and animals by produc in g var ious toxins. POIY- D- gluta mi c acid c ap sule and tox ins are enco ded by ge nes prese nt on two mega plasmids, de signated pX02 and pXO I, res pectiveli · 6 . Previous studi es have shown that 60-MDa plasmid pX0 2 is esse nti a l fo r caps ul e fo rmation and cap reg ion loca ted on the pl asmid enco des the ca ps ul ar protein. Isola ti on and identi fic ation of a nthr ax spores from env ironmental sa mple is co mpara ti vely dif ficult, while detection of B. anthracis from clini ca l specimens is simple though time consuming. Polymerase chain reac ti on ( PCR ) has been recently employed for detection of spores us in g primers s pecific to caps ul e or toxin ge nes ?· I). In the present inves ti ga ti on , we h ave reported a se nsi ti ve *Con'cspo nd ent auth or: E- ma il : drde @s ancharn et.in and specific PCR assay for detec ti on of B. anthracis spores from the environmental samples ( so il ). Th e method was used in co mbina ti on with a mo dified spore extrac ti on protoco l to facilitate DNA extr action fro m so il samples. Bacterial c ll ltures - Non path oge ni c B. allthracis sterne stra in was procur ed from In st itu te of Veterinary and Preve ntive Medicine, Ranipet, Ve ll ore ( In d ia). Spores of B. cereus and B. subtiiis were procured fro m Difco, USA. Spore prepara ti on of B. anthracis - B. athracis was grow n on Bra in Hea rt In fusion ( BHI ) aga r for 18- 24 hr at 37°C. Th e growth on the plate was scrapped and in oc ul ated into G-m e dium broth. Th e culture wa s allo we d to sporulate by incubating the broth at 30°C on a shaker at 200 rpm for four days as d esc ri bed by St ewart and Halvorson 5 . Sp ores were harvested by ce ntrifu ga ti on at 6000g fo r to min at 4°C and res us- pended in phosphate buffe r sa line (PBS, p H 7.2) be- fore heating at 60°C for 90 min to in ac ti vate any vegeta ti ve ce ll s. Pu rification of spores - Purification of B. an- thracis spores was carried out by 'aqu eo us polymer two phase system' as described by Sacks and Alder- ton 6 . Brie fl y, 3.4 ml of potassium phosphate buffer ( 3M, pH 7 . 1) wa s mixed with 1.1 2 g of po ly ethylene glyco l 4000 (PEG) and the final volume was made up to 10 ml w ith wate r including sample (spor es ). The mixture was homog enized vigo rously and ce ntri fu ged at 1500 g for 2 min in a swinging bucket rotor at room te mper a tur e. Th e upper phase co ntaining the spores wa s removed c arefully without disturbing th e intelf ace.