ŴƄţǀ źŤĩŚŝ ƶſ ƱŚƯżưƷ Ɔ ƽ ţ LjƳƺưƫŚſ ǀƠƾ ſŚŝ ſřźŤƳō ŽƺƬ ƹ
Transkrypt
ŴƄţǀ źŤĩŚŝ ƶſ ƱŚƯżưƷ Ɔ ƽ ţ LjƳƺưƫŚſ ǀƠƾ ſŚŝ ſřźŤƳō ŽƺƬ ƹ
ƱŚººŬƳŻƾĪºƃżěƭƺººƬƗƵŚĮººƄƳřŵƾººƄƷƹĦěƾºưƬƗƽƶººƬŬƯ ÎÍÔŚţÖКŚŰƠƇÎÐÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹ ƹŵ ŚŝžǀŤƀěŚǀƴǀſźƿƹžǀſřźŤƳōŽƺƬǀſŚŝƾƠǀţLjƳƺưƫŚſƽźŤĩŚŝƶſƱŚƯżưƷƆǀŴƄţ Źōƾ ſƾ ěžĪƬěƾŤƫƺƯƽŻřŶƳřƵřŹ ÎƽŶǀƗƭźĩřźŤƧŵÏƾƬƗŹƺěƶưƏŚƟÏƾƯźĩƾƬƗźŤƧŵÎƾƳŚƃƹźƟƽźƠƇžĭźƳ [email protected]ƾƫƺĪƫƺƯƽĥƺƫƺǀŝšŚƤǀƤŰţżĩźƯŪƗřƶǀƤŝƾĪƃżěƭƺƬƗƵŚĮƄƳřŵƱřźƸţƩƹƺƀƯƽƵŶƴƀ ƿƺƳ ÖÎÎÎÏÖƁźƿŸěÖÎÔÎÏŢƟŚƿŹŵ ƵŶǀĪģ ƆǀŴƄºţƹŶƳƺºƃƾ ƯśƺƀŰƯƱŚƀƳřƽřźŝƾƘƣřƹƽŚƷƱĥƺţŚěŻřžǀŤƀěŚǀƴǀſźƿƹžǀſřźŤƳōŽƺƬǀſŚŝƾƠǀţLjƳƺưƫŚſƽźŤĩŚŝƶſƝŶƷƹƶƴǀƯŻ žǀŤƀºěŚǀƴǀſźƿƹƮųŻƵŚǀſŦƗŚŝžǀſřźŤƳōŽƺƬǀſŚŝƶŞƈůƽŹŚưǀŝŦƗŚŝƾƠǀţLjƳƺưƫŚſŢſřƽŹŚǀƀŝŢǀưƷřżŗŚůŚƷƱōƭŚĮƴƷŵƹŻƹƖƣƺƯƶŝ ŚƷƽ źŤĩŚŝƲƿřƆǀŴƄţĨǀſLjĩƽŚƷƁƹŹ ŵŵźĭƾ ƯƵŵŚƠŤſřżǀƳƾƯŚƔƳĨƿĥƺƫƺǀŝƪƯřƺƗƱřƺƴƗƶŝƽźŤĩŚŝƕƺƳƶſƲƿřŵƺƃƾ ƯƱƺƗŚƏƽŹŚưǀŝŜŞſ ĨºƿŹŵƽźŤĩŚºŝƶºſƲºƿřƱŚºƯżưƷƆǀŴƄºţƽřźŝƖƿźſƹŶƿŶūƾƫƺĪƫƺƯƾƈǀŴƄţƁƹŹĨƿƽƶƿřŹřźƋŚůƽƶƘƫŚƐƯ ŢſřƵŶǀĤǀěƹƾƳLJƺƏ ŶƃŚŝƾ ƯŹōƾ ſƾ ěžĪƬěƾŤƫƺƯƂƴĩřƹ LjƳƺưƫŚºſinvA,hpžƳLJƹźºƿƹƽŚºƷƱĥ ƽƹŹźºŝPolymerase Chain Reaction, PCR)ŻřźºƯƾºƬěƾƿřƵźǀŬƳŻƂƴĩřƹƾſŹźŝƁƹŹ ƾƯƺºƳĥ DNAŶƃƭŚŬƳřƹƾůřźƏŹōƾſƾěžĪƬěƾŤƫƺƯƹžĪƬěƾƳƺƿšŹƺƇƶŝžǀŤƀěŚǀƴǀſźƿplaƹžǀſřźŤƳōŽƺƬǀſŚŝPa,chr ƾƠǀţ ŢºƣŹƹƾºƴƬĩƁŹŚưƃƁƹŹƹŵŻřƂƴĩřƹŢǀſŚƀůƾſŹźŝŢƸūŶƃƵŵŚƠŤſřPCRƂƿŚƯŻōƹźưƿřźěƾĭĦƿƹƾſŹźŝŢƸūŚƷƽ źŤĩŚŝźƿŚſŻř ŶƃƵŵŚƠŤſřƭƺƳĥ ŶºƿŵźĭŶºǀƫƺţźºƔƳŵŹƺºƯƽƵŻřŶºƳřŚŝšLJƺƈŰƯƹŢſřƾƇŚƈŤųřŚƷźưƿřźěƶĩŵƺưƳƆŴƄƯŵŹřŶƳŚŤſřƽŚƷƶƿƺſŚŝƁƹŹƲƿřƾſŹźŝŚƷƶŤƟŚ ƿ ŹƺºƏƶºŝŚºƷƽ źŤĩŚºŝƲºƿřŻřƭřŶºĩźºƷƽřźºŝƾƴƬĩŶůřƹÎÍŚţÎŚƿƹƭƺƳĥƾĜĩÎÍŚţÎŢſřŹŵŚƣƁƹŹƲƿřƶĩŵřŵƱŚƄƳƁƹŹŢǀſŚƀůƾſŹźŝ ƹŶƳŶƃƝŵřźţƲǀǀƘţžĩřźŤƳōƪǀſŚŝÎÍÕÐbpƩƺƈŰƯżūƶŝšŚƘƐƣƭŚưţPCRŹŵƵŶƃŵŚŬƿřšLJƺƈŰƯŶǀƿŚţƽřźŝŶƷŵƆǀŴƄţƾƇŚƈŤųř ŶƿŵźĭŶǀƿŚţƾưƿżƳōƁźŝÎÏÒźưƿřźěŚŝžĩřźŤƳō ƽŚºƷƽ źŤĩŚºŝźƿŚſŻřŚƷƽ źŤĩŚŝƲƿřƆǀŴƄţƹƺŬŤƀūƽřźŝƶƴƿżƷƮĩƹƖƿźſƢǀƣŵƽŹřżŝřƵŶƃƾůřźƏƁƹŹƶƘƫŚƐƯƲƿřŽŚſřźŝƽźǀĭƶŬ ǀŤƳ Ŷƴĩƾ ƯƮƷřźƟƾŤƀƿŹƹźţƺǀŝŵŹřƺƯŹŵřŹŚƷƽ źŤĩŚŝƲƿřƆǀŴƄţƹƺŬŤƀūƹƞƬŤŴƯƽŚƷƶƳƺưƳŹŵƪƯŚƗƶſƲ ƿřƖƿźſƆǀŴƄţƹƵŵřƺƳŚųƮƷ Źōƾ ſƾ ěžĪƬěƾŤƫƺƯžǀŤƀěŚǀƴǀſźƿƾƠǀţLjƳƺưƫŚſžǀſřźŤƳōŽƺƬǀſŚŝƆǀŴƄţƽŶǀƬƧƱŚĭĥřƹ ƽřŻƽ ŹŚºưǀŝƹŶƳŹřŵƱŚƀƳřƾƿřŻƽ ŹŚưǀŝƽřźŝƾƿLJŚŝƪǀƀƳŚŤěƶĩ řżƿŹŚºưǀŝƪºƯřƺƗƲºƿřŚŝƶƬŝŚƤƯƽřźŝŶƴŤƀƷƱŚƀƳřƽřźŝƾƘƣřƹ ƶƯŶƤƯ žǀŤƀºěŚǀƴǀºſźƿƹžǀºſřźŤƳōŽƺƬǀºſŚŝƾºƠǀţLjƳƺưƫŚſ ŶƳƺƃƾ ƯƶŤųŚƴƃ řŻƽ ŹŚưǀŝƽŚƷƽ źŤĩŚŝAƵƹźĭƪƯřƺƗƱřƺƴƗƶŝ ƱřźƸţšŚƤǀƤŰţƭƺƬƗŶůřƹŵřŻōƵŚĮƄƳřŵƭƺƬƗƵŶĪƄƳřŵƾƫƺƨƫƺƯƾƫƺƬſŶƃŹřŽŚƴƃŹŚĩÎ ƱřźƸţšŚƤǀƤŰţƭƺƬƗŵřŻōƵŚĮƄƳřŵŹŚǀƄƳřŵƽĥƺƫƺƿżǀƟƾƈƈŴţƽřźŤĩŵÑ ŪƗĽřƶǀƤŝƾĪƃżěƭƺƬƗƵŚĮƄƳřŵƾƫƺĪƫƺƯƽĥƺƫƺǀŝšŚƤǀƤŰţżĩźƯŵŚŤſřƾƫƺĪƫƺƯƽĥƺƫƺǀŝƾƈƈŴţƽřźŤĩŵÏ ĽřƶǀƤŝƾĪƃżěƭƺƬƗƵŚĮƄƳřŵƾƫƺĪƫƺƯƽĥƺƫƺǀŝ šŚƤǀƤŰţżĩźƯƾƷŚĮƄƿŚƯŻōƭƺƬƗŽŚƴƃŹŚĩÐ Źōƾſ ƾěžĪƬěƾŤƫ ƺƯƽŻřŶƳřƵřŹŚŝƱŚƯżưƷƆǀŴƄţ 94 ŢƴºſŹƺƬƟƺƳƺưƿřƹřżºƿLJřƱƺºģƽźºĮƿŵšŚƄºƿŚƯŻōƲǀƴĤưƷ ŢºſřŹřŵŹƺºųźŝƽŹŚǀƀºŝŢǀưƷřŻřƖƿźſƱŚƯŹŵƹƆǀŴƄţ ƽřŻƽ ŹŚºưǀŝźǀƛ ƽŚºƷƪ ǀºſŚŝƶºŝŹŚǀƀºŝŢƷŚŞºƃƪǀƫŵƶŝƮųŻ ÓÍÍŵƹŶºůŜ޺ſƹLjŤŞƯƽŹŚưǀŝƲƿřƶŝƱŚƸūŹŵźƠƳƱƺǀƬǀƯ ƵŚǀºſƪºƯŚƗƾƿŚºſŚƴƃƹƆǀŴƄºţÖƹÎÍŵŵźĭƾ ƯƵŵŚƠŤſř ƽŚƷƵŚĮƄ ƿŚƯŻōŚƸƴţƹŢſřźǀĭŢƣƹƹƪĪƄƯħŚųŹŵŵƺūƺƯ ƲǀƗŹŵƹŶƴƃŚŝƾ ƯƱōƢǀƣŵ ƾƿŚſŚƴƃƶŝŹŵŚƣƖūźƯƹƶŤƟźƄǀě ƱřƺººǀůŹŵšŶºººůƲǀºººǀƘţƩƹřŶºººŤƯƽŚºººƷƁƹŹŹŵƩŚºººů ƶºŝŻŚǀƳƱŵƺŝźǀĭŢƣƹźŝƵƹLjƗƶĩŢſřƽŹƹźƋƾƷŚĮƄƿŚƯŻō ƪºƯřƺƗŚºŝŹŚºĩƅŚºų ƾŤƀºƿŻ ƾºƴưƿřšŚºƳŚĪƯřƹšŚƀǀſŚţ ŚºŝƶĩƽŵřźƟřƽřźŝřŹƾţřźƐųƹšLjĪƄƯƹŵŹřŵřŹħŚƳźƐų ƆǀŴƄºţŹŵÎÎŵŻŚºſƾ ºƯƮƷřźºƟ Ŷºƴƴĩƾ ƯŹŚĩƪƯŚƗƲƿř ƾººſŹźŝƾƿŚǀưǀººƃƺǀŝƽŚººƷŢƀººţƭŚººŬƳřŻřƱƺƗŚººƏƪººƯŚƗ šŚººƳřƺǀůƶººŝŮ ǀººƤƬţƾƇŚƈººŤųřĥŚººƟƺƿźŤĩŚŝƶººŝŢǀſŚƀººů ƵŵŚƠŤººſřƾƇŚƈººŤųřƽŚººƷƎ ǀº ŰƯŹŵŢƄººĩƹƾƷŚĮƄº ƿŚƯŻō ŜºſŚƴƯƽŚºƷƁƹŹƱŵƺŞƳŽźŤſŵŹŵźĮƿŵƝźƏŻřŵƺƃƾ Ư ƲºĪưƯƾƈǀŴƄţżĩřźƯƶŝƵŵƺƫōƢƏŚƴƯŻřŚƷƶƳƺưƳƩŚƤŤƳř ƽřźŝ īźºƯŚºƿ ŚºƷƶºƳƺưƳ ƾĭŵƺºƫōŚºƷƱōƱŶƃĨƄųŦƗŚŝŢſř ƽźŤĩŚºŝŢƄºĩƱƺģƩƺưƘƯƽŚƷƁƹŹ ÎÏŵŵźĭŚƷƽ źŤĩŚŝ ƽźŤĩŚºŝƆǀŴƄºţ ƽřźºŝŪºƿřŹ ƽŚƷƁƹŹ Żř ƾƿLjƏŵŹřŶƳŚŤſř ƹźºǀĭŢºƣƹĨºƿĥƺƫƺƿźŤĩŚŝŪƿřŹ ƽŚƷƁƹŹƲƿřƭŚưţ ŶƃŚŝƾ Ư ƪººƯŚƗƢºǀƣŵƹƖƿźººſƆǀŴƄººţŢººƸūŹŵƵŵŚƠŤººſřƪººŝŚƣźǀƛ ƾƫƺººĪƫƺƯƽŚººƷƁƹŹŹŵƶººĩƾƫŚººůŹŵÎÐŢººſřƽŹŚººưǀŝ ŹŵƮǀƤŤƀºƯŹƺºƏƶºŝŚºƷƾ ſŹźŝƶƳƺĭƲƿřƶŝŻŚǀƳƱƹŶŝƱřƺţƾ Ư ƱLJƹźºƿƹƾƳƺºƠƗƪºƯŚƗƾƿŚºſŚƴƃƶºŝƭřŶƣřƵŚţƺĩŹŚǀƀŝƾƳŚƯŻ ƹĨºƿĥƺƫƺǀŝšLjºưůŹŵƩƹřŶºŤƯ ƽŚºƷƁƹŹ ƲƿřźŝŚƴŝ ŵƺưƳ ƾƈºǀŴƄţƽŚºƷƁƹŹƶŝŻŚǀƳƹƵŵƺŝƁŻŹřŶƣŚƟƾŤƀƿŹƹźţƺǀŝ šŚƯřŶºƣřƱřƺºŤŝƶºĩŶºƃŚŝƾ ºƯŢƗŚºſŶƴģŻřźŤưĩ ŹŵƖƿźſ ƲºƿřƹŵƺºưƳƩŚºưƗřřŹ ƽŹŚºưǀŝƕƺǀƃ Żř ƽźǀĮƄǀěƹ ƾƳŚƯŹŵ ƾºŞƏƆǀŴƄºţ ƽŚƷƵŚĮƄ ƿŚƯŻōŹŵ ƾƿřƵŵźŤ ƀĭŵźŝŹŚĩŚƷƁƹŹ ŜŞſĨƿĥƺƫƺǀŝƪƯřƺƗƖƿźſƆǀŴƄţƾƿŚƳřƺţƱřŶƤƟŵŹřŵżǀƳ šřŶºƿŶƸţƹšŚƯŶºƇƱřżǀƯźƔƳŻřƪƯŚƗƲƿřƶĩŢſřƵŶƿŵźĭ ƕƺǀºƃƪºǀƫŵƶºŝźţħŚƳźƐ ųƹ ƾƿřƶŤƀƷ ƽŚƷŭLjſŚŝĬƴſƮƷ ÎÓŻřƂǀºŝƶºĩŢºſřŶºǀŗƺƠǀţŚƿƶŞƈůƪƯŚƗƾƠǀţLjƳƺưƫŚſ ƽŚƴŨŤºſřƶºŝŚºƷLjƳƺưƫŚſƽƶưƷÎŶƃŚŝƾ ƯīźƯŵŹƺƯŹřżƷ ƹƱŚƀººƳřŹŵƾººƿřŻƽ ŹŚººưǀŝŜ޺ºſƎººƤƟƶººĩƾº ƠǀţLjƳƺưƫŚººſ ƱŚĭŶºƳźěƹƱřŹřŶƳŚŤƀºěƱŚƀƳřƽřźŝŶƃŚŝƾ ƯƾƫŚƗƽŚƷƾ ŤƀŴƳ śōƢºƿźƏŻřƱōŢƿřźºſŢºƸūƲǀºưƷƶŝŶƃŚŝƾ ƯřŻƽ ŹŚưǀŝ ŹŚºưǀŝƱŚĭŶƳźěƹƱřŹřŶƳŚŤƀěƱŚƀƳřŹřŹŵřƹƕƺƟŶƯƾƿřŸƛŵřƺƯ ƾƳƺºƠƗƽŹŚºưǀŝƲŝŹŚºƃŚºƿƮºųŻƵŚǀºſÏŶƃŚŝƾ ƯƵŵƺƫōƹ žǀºſřźŤƳōŽƺƬǀºſŚŝƎſƺţƶĩŢſřƭřŵƹƱŚƀƳřƲǀŝħźŤƄƯ ƵŶƷŚƄºƯƱŚƸūƍŚƤƳƭŚưţŹŵƾƯřŵ ƽŹŚưǀŝƲƿřŵŵźĭƾ ƯŵŚŬƿř ƾºƯřŵšLJƺƈºŰƯŻřƮǀƠŤƀºƯźºǀƛŹƺºƏƶŝ ƽźŤĩŚŝƹŵŵźĭƾ Ư ƱŚƀƳřƶŝƮųŻƵŚǀſƪǀſŚŝŹƺĜſřƎſƺţƮǀƤŤƀƯŹƺƏƶŝŚƿ Ƶŵƺƫō ƽƺºƿŹƾŤºſƺěƪĪºƃŹŚºƸģƶºŝƮºųŻƵŚǀºſŵƺƃƾ ƯƪƤŤƴƯ ƪº ǀƫŵƶººŝƽźŤĩŚººŝƲº ƿřŵƺººƃƾ º ƯƵŶº ƿŵŢº ƿĦƴƴƯƹƾº ƃŹřƺĭ LJŚºŝšŶºůŹƺĜſřƲŤƃřŵŻřšŹŚŞƗƶĩƱōƵĦƿƹšŚǀƇƺƈų ħŚºƳźƐųƪºƯřƺƗŹŶºƇŹŵƽźǀĭŹŚºĩƶºŝƹƵƺŞƳřŶǀƫƺţŢƫƺƸſ ƱƺƗŚºƏÐŵŹřŵŹřźºƣƮƀºƿŹƹźţƺǀŝƹĨºƿĥƺƫƺǀŝ ƽŚƷŭLjſ ƽźŤĩŚŝŚŝƾĭŵƺƫōƩŚŞƳŵƶŝƶĩŢſř ƾĩŚƳźƐųƹ ĨƬƸƯ ƽŹŚưǀŝ Ñŵƺƃƾ ƯŵŚŬƿřƱŚƀƳřƹƱŚĭŶƳƺūŜƬƛřŹŵžǀŤƀěŚǀƴǀſźƿ ŹƺĜºſřƹŢºĩźůŶºƣŚƟƾºƠƴƯƭźºĭƪǀºſŚŝƺĩƺĩ ƽźŤĩŚŝƲƿř ƲǀºŝźºǀĭƶºưƷƾºƋźƯƪĪºƃƶºŝƩƺºưƘƯŹƺºƏƶŝƶĩŶƃŚŝƾ Ư Żř ƽŹŚºưǀŝƪºƯŚƗÒŵƺºƃƾ ºƯƪƤŤƴƯƁƺƯŶƴƳŚƯ ƾƳŚĭŶƳƺū ƪºŝŚƣƱŚƀƳřƶŝĨĩƵĦƿƹƶŝƵŵƺƫō ƽŚƷŢ ƿŻřŹŚěƺŤĩřƁżĭƢƿźƏ ƾĩŹŚºǀųƾƴǀƫŚºŝƪĪºƃƶºſƶºŝƽŹŚºưǀŝÓƹÔŢſřƩŚƤŤƳř ŢƫŚůƲƿźţŶƿŶƃ ƾƀƠƴţƭźƟŵƺƃƾ ƯźƷŚƓ ƾƀƠƴţƾưǀƀŤĜſ ƲƿźŤƄºǀŝƽŹŚºưǀŝŻřŢƫŚůƲƿřŶƴĩƾ ƯŵŚŬƿřřŹƽŹŚưǀŝƾƴǀƫŚŝ ƱŚƀƳřƶŝƱŚƀƳřŻřƾŤůřŹƶŝƹŵŹřŵƵřźưƷƶŝřŹźǀƯƹīźƯŵŹřƺƯ ƽźŤĩŚºŝƾƷŚĮƄºƿŚƯŻōƆǀŴƄºţƽřźºŝÕŢºſřƩŚƤŤƳřƪŝŚƣ żºƜƯƱƺºųŢƄºĩŻřƾºĪǀƴǀƬĩ ƽŚºƷƶºƳƺưƳŹŵƶŞƈºůƪƯŚƗ ƹŹřŹŵřŢƄººĩƹƕƺƟŶººƯŢƄººĩƹƩŚººŤĩŹęřƺººſƱřƺŴŤººſř ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ 95 ƱřŹŚƨưƷƹƾƳŚƃƹźƟƽźƠƇžĭźƳ ŢĩźºƃƎºſƺţƹƾůřźƏ pla ƱƺƗŚƏƪƯŚƗ ƽźŤĩŚŝ ƽƵŶƴƷŵ DNASISƽŚºƷŹřżƟřƭźºƳŻřƵŵŚƠŤºſřŚŝƹŶƃ ƶŤųŚſƱĥŚƴǀſ ÓÔÎBlast HITACHI,TOKYO,japon ÎÑŶººƃŚŝƾƳŚººƸūƽźººǀĭƶº ưƷƾººŤůƹźººĮƿŵƢƏŚººƴƯƶººŝ źƿŚººſŚººŝŢƷŚŞººƃƹŹŚŤųŚººſźººƔƳŻřOLIGOƹVersion ƾſŹźŝƁƹŹ žººƳřźƟŹƵŚĮººƄºƿŚƯŻōŻřŵŹřŶººƳŚŤſřšŹƺºƇƶŝ ŚƷƶƿƺſ źºǀŨĪţƽƶºƘƐƣƵŻřŶºƳřƹƭƺƳĥ ƽƹŹźŝŚƷźưƿřźěƱŚĪƯƲǀƴĤưƷ ƱLJƹźºƿƹźºǀƛƁƺºſŻřƱLJƹźºƿƹƽƶƿƺºſŚºŝƽŹŚĩšřźƐų ŶºƃƶŤºƃƺƳƢºǀƤŰţƲºƿřŹŵƵŶƃƵŵŚƠŤſřƽŚƷźưƿřźěžƳŚĪſ ŚºŝƾţƹŚºƠţĢǀºƷƾºſŚƴƃśźºĪǀƯźºƔƳŻřƶºĩŶƿŵźĭƵŵŚƠŤſř ƩƺºƯƺĪǀěÎÍÍŢºƔƬƛŚŝƾƤƿŹżţźƐƤƯśōŹŵźưƿřźěƵŶƳŻŚſ ƽŚºūƶºŝƾºƴưƿřŢºƸūƶºŝƹŢºſřƱLJƹźºƿƹƽŚƷŶǀưǀſLjě ŶƃƽŹřŶƸĮƳŵřźĭƾŤƳŚſƽƶūŹŵ ÏÍƽŚƸƴƯŹŵƵŵŚƠŤſřŢƸū ƱřźºƿřƱŚŤºſŵźĩŻřƵŶºƃřŶºūƽƶƿƺſžǀŤƀěŚǀƴǀſźƿƽźŤĩŚŝ ƽƶºūŹŵÐÔŹŵƹŮǀºƤƬţŚºƿŹƺƫƖƿŚºƯƎǀºŰƯƹŶºƯŚūŢƄĩ ƱřƺƴƗƶŝƶĩƾƿŚƷƶƳƺưƳ ŢƃřŶƸŝšŹřŻƹžƳřźƟŹƵŚĮƄƿŚƯŻōŻř ƅƺƈººųƶººŝƹŹŚŤųŚººſƹƪĪººƃźººƔƳŻřŚººƷƾ ƳƺººƬĩƾºſŹźŝ ƵŵřŵŮǀƋƺţÎ ƩƹŶūŹŵŚƷƱōƶƿƺſ ƽƵŹŚưƃƹŢƀǀƫŶƿŵźĭ DNAƖƿŚƯŢƄĩŻřźŤǀƫƾ ƬǀƯÒŚţÎÒŻřžĜſŢƟźĭƭŚŬƳř ŹŵŶƿŵźĭƶǀƸţ ƱĥŚƴǀſŢĩźƃ Żř Taq ƮƿżƳōƹŚƷŶǀţƺŘƬĩƺƳ Żřƾºſƾ ſÎÒƶĩŶƃƭŚŬƳřšŹƺƇƲƿřƶŝ DNA ũřźŴŤſř ƵŵŚƠŤºſřƾºţŹřźůƽŚºƷƶºųźģŢƸūƱŚưƫōƝŹƹŶƴěřŢĩźƃ ĥƺƠƿźŤƳŚºſÒÍÍÍrpmŹƹŵŹŵƶºƤǀƣŵÒŚţКŶƯƶŝƹƶŤŴƿŹ TBEXÍÒźƟŚººŝŚººŝƱōƶººƿŸƜţƖººŞƴƯƹŶƸƄººƯƂƷƹĦººě ƶƤǀƣŵÎÍšŶƯƶŝƹƵŵźĩƍƺƬŴƯLjƯŚĩƵŵźĩƶƟŚƋřSTEźƟŚŝ ŶºƃƵŵŚƠŤºſř žǀƬĮƳř UVItec ŢĩźƃŢųŚſUVIdoc řŹƩƺººƬŰƯƹƵŵźººĩƶƟŚººƋřƱƺǀƀººƳŚĜſƺſƶººŝSDSŶººƇŹŵ ŶƃƵŵŚƠŤſřŻŚŝŢƠūÎÍÍÏÍÍÐÍÍÑÍÍÒÍÍÎÍÍÍ ÎÓŶºƿŵźĭśŚºŴŤƳřƹ ƾºſŹźŝƶŝŚƄºƯ ƽŚºƷƮƀ ǀƳŚĭŹřƹźĪǀƯ ƪºǀƫŵƶºŝŶºƃƶºǀƸţƱřźºƸţŹƺŤſŚěƺŤǀŤƀƳřƹŢƃřŶƸŝšŹřŻƹ ÐƩƹŶºūŹŵŶºƃƵŵřŵŮǀƋƺţÏ ƩƹŶū ŹŵƝŶƷƱĥƹƶŤƟŚƿ ƾºƯřŵƲƀĩřƹ ƽƶƿƺſŚƿ ƱźŤƃřƶŝƭƺſƺƯžǀſřźŤƳōŽƺƬǀſŚŝ ŢĩźºƃƎºſƺţƵŶºƃźĩŷŢƔƬƛŽŚſřźŝƾŤƟŚƿŹŵƽŚƷźưƿřźě ŻřƾĪƿŶƣŚƟŚƟźƇƾƫƺĪƫƺƯźƔƳŻřƾƫƹŵŹřŶƳƾƄůƹƽƶƿƺſ ƹƶºǀƸţŢºƔƬƛƱōŻřƩƺºƯƺĪǀěÏÍŢºƣŹžĜºſƹƵŶƃƶǀƸţ ŵƺƃƾ ƯƵŵŚƠŤſřƱōŻřƾƈǀŴƄţšŚƄƿŚƯŻōŹŵƾƄůƹƽƶƿƺſ ƎǀºŰƯŹŵƵŶºƃƶºǀƸţ ƽŚƷƽ źŤĩŚŝDNAũřźŴŤſřƹŢƄĩ ƵŶƃƶǀƸţŹŚưǀŝŻřƵŶƃřŶūƾƠǀţLjƳƺưƫŚſƹKIMƶŝƭƺſƺƯ źºŝƵƹLjºƗŶºƃŢƄºĩŢƗŚºſÏÑŚºţÎÕšŶƯƶŝŵřźĭƾŤƳŚſ ƶǀƸţƱřźƿřŹƺŤſŚěƺŤǀŤƀƳřƵŚĮƄƿŚƯŻōŻřŶƃƵŵŚƠŤſř ƾƠƴƯƩźŤƴĩ żºǀƳƾƿŚǀưºƃƺǀŝƽŚƷŢƀţƭźĭ ƽżǀƯōĬƳŹƾƴƬĩƽŚƷƾ ĭĦƿƹ źƟŚºŝMgClÏħźºƯŢĩźºƃŻřƾƿŚǀưǀƃŵřƺƯŢſřƵŶƃ ƁƹŹŶºƿŵźĭũřźŴŤºſřÎÒƩƹřŶºŤƯƽŚºƷƁƹŹƶºŝ ƾƯƺƳĥ ŢųŚºſMastercycler Gradient ƵŚĮŤſŵŻřƢǀƤŰţƲƿř ƝŹƹŶƴěřƽƶƫƺƫƪųřŵřŹLB ƎǀŰƯŹŵ ŚƷƽ źŤĩŚŝ ƶƳŚŞƃ ŢƄĩ ŚºƿŚěŢĩźºƃŢųŚſĨģƺĩƾƤƟřŻŹƺƟƹźŤĪƫřƵŚĮŤſŵŻřŶƃ ŻřřŶºƳLJ ÏÍÍ śƺſŹ ƶŝƹƶŤŴƿŹŹƹŵřŹ ƾƿƹŹƩƺƬŰƯŶƿŵźĭ ƵŚĮŤºſŵŻřƩĥƾºſŹźŝŢºƸū ŶƃƵŵŚƠŤſřŻŹƺƟƹźŤĪƫřŢƸū ÏƵŶºƴƴĩżǀƫźƟŚŝźŤǀƫƹźĩŚƯÏÍÍƮǀƷŵƾ ƯŹřźƣơŚţřšŹřźůŹŵ ŶºƳŚŝƪƯŚºƃŻŚºŤƴƯźƟŢĩźºƃŻŚºŝŢºƠūÎÍÍƾƫƺĪƫƺƯźĩŹŚƯ ƮƿŶºſšŚŤºſřźŤǀƫƹźĩŚƯÏÍÍƮƿŵřŵƱŚĪţžĪţŹƹŚŝšŶƃƶŝ ƂŴŝƹŵƞƿŵŹŽŚſřźŝ ƾƠǀţLjƳƺưƫŚſ ƽřźŝźưƿřźěƾůřźƏ ƖƿŚºƯŻřřŹśƺºſŹ ÎÍÍÍÍrpmŹƹŵŹŵŹřŶƫŚĤŴƿĥƺƠƿźŤƳŚſ ƾſŚſřƂŴŝƹŵŽŚſřźŝžĩřźŤƳōƽřźŝhpƹ invA ƝŶƷ ƩƺºƬŰƯƮŬůƮƷŶƃ ƪƤŤƴƯƵŻŚţ ƽƶƫƺƫƶŝ Ţſř ƾƯƹŻƺƯƹźĩ ƪǀĪƄºţƭƺºƳĥƾºſŚſřƂºŴŝĨºƿŽŚºſřźŝ žǀŤƀěŚǀƴǀſźƿ ŚºŝƶºƤǀƣŵÒšŶƯƶŝžĜſƹƵŵřŵƱŚĪţŹŚŝÎÍƹƶƟŚƋřƶƫƺƫƶŝ ƱĥŽŚºſřźŝŢºƤǀƤůŹŵƾƠǀţLjƳƺưƫŚſƽźŤĩŚŝƭƺƳĥŻřƾſŚſř DNAƽƹŚºůƶºĩƾºƿƹŹƩƺºƬŰƯƮƿŵźºĩřŶūƾƿƹŹƝŚƠƃ ƽřźººŝchrƹpAžĩřźººŤƳōƪǀºſŚŝƭƺººƳĥƽƵŶººƴƷŵƪǀĪƄººţ ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ Źōƾſ ƾěžĪƬěƾŤƫ ƺƯƽŻřŶƳřƵřŹŚŝƱŚƯżưƷƆǀŴƄţ 96 chrƹpAƾº ƠǀţLjƳƺưƫŚººſhpinvAƽŚººƷƱĥ ƽŚººƷźưƿřźě ƶƤǀƣŵÏÍšŶƯƶŝřŹƶƫƺƫƹƶƟŚƋřƪƳŚěƹźěƹżƿřƵŶƃřŶūƾƿƹŹ ƵŶºƃũřźŴŤſřƭƺƳĥƽƹŹźŝơƺƟƎƿřźƃŚŝPCRƂƴĩřƹźƔƳ rpmÎÒÍÍÍŚţÎÍÍÍÍŹŵƹƶūŹŵÑƽŚƯŵŹŵƶƤǀƣŵÎÒšŶƯ ŵŹƺºƯƽŚºƷƽ źŤĩŚºŝƆǀŴƄţŢƸūƹŚǀƴǀſźƿplaƹžĩřźŤƳō ŶƃƭŚŬƳřƾƠƴƯƩźŤƴĩƽŚƸƿźŤĩŚŝ ƹŵŻřPCR ŢǀſŚƀůƱřżǀƯ ƾſŹźŝŢƸūŢǀſŚƀůƲǀǀƘţ ŶƃƵŵŚƠŤſřDNAŢƔƬƛƹƾƳƺƬĩƁŹŚưƃƁƹŹ ƩƺƈºŰƯƾºưƿżƳōƁźºŝƁƹŹƹŵƶºŝPCR ƩƺƈŰƯŶǀƿŚţ ƭŚºŬƳřĬƴǀƀºƳŚĪſPCRƩƺƈºŰƯƞºƿŵŹƲǀǀƘţƹPCR ŢƿŚººſŶūřƹƶººĩžĩřźººŤƳōƪǀººſŚŝÎÍÕÐbpƩƺƈººŰƯŶººƃ ƪºƿŷƁƹŹƶŝƾưƿżƳřƁźŝƢƿźƏŻřŶƃŚŝƾ ƯHindIII Ɓźŝ ƶºŝřŹƶºƫƺƫžĜºſƮƿŵźºĩƶŝƺƨƳřŵřźĭƾŤƳŚſƽƶūŹŵÏÍŹŵ ŹƹŵřŹƾººƿƹŹƩƺººƬŰƯĥƺƠƿźŤƳŚººſŻřŶººƘŝƮƿŵźºĩĥƺƠƿźŤƳŚººſ ƶºƤǀƣŵÐÍŚţÏÍšŶƯƶŝśƺſŹƱŶƃĨƄųŻřŶƘŝƹƶŤŴƿŹ źºƐƤƯśōřŶºƳLJÎÍÍŹŵŵřźĭƾ ŤƳŚſƽƶūŹŵÓÒŚţÓÍŹƺƟŹŵ ÖÒƽŚºƯŵŹŵƶºƤǀƣŵÎÍšŶºƯƶºŝřŹƩƺºƬŰƯŶƃƪůƪƿźŤſř ÏšŶºƯƶºŝřŹƶºƫƺƫžĜſƮƿŵřŵšŹřźů ŵřźĭƾŤƳŚſƽƶūŹŵ ƝŚƠºƃƩƺºƬŰƯŻřƹƵŵźºĩĥƺƠƿźŤƳŚºſ rpm ÎÏÍÍÍŹŵƶƤǀƣŵ ÎÒŶƿŵźĭƵŵŚƠŤſřPCRŢƸūřŶƳLJÒƾƿƹŹ ƮƿżƳōźŤǀƫƹźĪǀƯÎƽƹŚůƽƶƫƺƫƶŝƶĩšŹƺƇƲƿřƶŝŶƃŶǀƿŚţ ƾŝŚºƿŻŹřŢºƸūPCR ƂƴºĩřƹPCR žºĪƬě ƾŤƫƺƯƭŚŬƳř źºŤǀƫƹźĪǀƯÏÍƾƿŚƸƳƮŬůƶŝƪƿźŤſřźƐƤƯśōŚŝƹƵŶƃƶƟŚƋř ƾƯƺºƳĥƽƶºƳƺưƳŻřƩƺƯƺĪǀěÎŹřŶƤƯŶƃƭŚŬƳřźŤǀƫƹźĪǀƯÏÒ PCRƩƺƈºŰƯŻřźŤǀƫƹźĪǀƯÒŹřŶƤƯƮƿżƳōźƟŚŝƹHindIII ƽƶºūŹŵÐÔƽŚºƯŵŹŵŢƗŚºſÏšŶƯƶŝƍƺƬŴƯƲƿřƹŶǀſŹ ƩĥŹŵƱōŻřźºŤǀƫƹźĪǀƯÎÍžĜºſƹƶºŤƟźĭŹřźºƣŵřźºĭƾ ŤƳŚſ ŚºŝƵřźưƷƵŶƃźǀŨĪţšLJƺƈŰƯ ƽƶǀƤŝƹŶƿŵźĭ ƾſŹźŝŻŹŚĭō ŢĩźºƃƶºŝĬƴǀƀƳŚĪſƽřźŝŚƷƱōƾŞƤƗƹ ƾƿƺƬū ƽŚƷźưƿřźě ŶƿŵźĭƩŚſŹřƶƏƺŝźƯ ŻřƪºƇŚůžƳŚĪºſƪƯŚƃ ƾŤƟŚƿŹŵ ƽŚƷƞ ƿŵŹžƳŚĪſżǀƫŚƳō ƮºƷŚºŝ ƾƳŚƃƺěƮƷƹŢƣŵźƔƳŻřƶƘƐƣźƷ RƹFƽŚƷźưƿřźě ŵƺºŝŹŚƔŤƳřšŚƘƐƣƽƵŻřŶƳřƾƷŚţƺĩƶŝƶūƺţŚŝŶƿŵźĭ ƾſŹźŝ ƾƿŚºſŚƴƃ ƽřźŝŶƃŚŝƶŤƃřŵŵƺūƹ ƾƳŚƃƺěƮƷ ƽŵŚƿŻŹřŶƤƯƶĩ ŢĩźºƃŢųŚºſDNASIS VerÏÓ ƽƶƯŚƳźŝŻř ƾƳŚƃƺěƮƷ žƳŚĪºſ ƽŹŚŤºƃƺƳƪºƿŚƟƶĩƪĪƃƲƿŶŝŶƃƵŵŚƠŤſř ƾģŚŤǀƷ RźºưƿřźěžƳŚĪºſReverse Complement ŚŝFźưƿřźě ƹƪƈŤƯƮƷƶŝALIGNƶƯŚƳźŝŻřƵŵŚƠŤſřŚŝƹŶƿŵźĭƶƀƿŚƤƯ ŶºƃƪºƇŚůƶºƘƐƣźºƷƽřźºŝƾƬºƇřžƳŚĪſƱōƂƿřźƿƹŚŝ ĨºƳŚŝŚŝÎÓBLASTÏÏÖƽƶƯ ŚƳźŝŚŝƪƇŚůƽŚƷžƳŚĪſ ƶºƯŚƳźŝŻřŶƿŵźĭ ƾƿŚſŚƴƃŚƷšƹŚƤţƹŚƷŢƷŚŞƃƹƶƀƿŚƤƯƱĥ ƲºƿřŻřƪºƇŚůžƳŚĪºſƹŵƽƶƀ ºƿŚƤƯŢƸūżǀƳDNASIS ƹŢƷŚŞººƃƱřżººǀƯƾƿŚººſŚƴƃƹƖººūźƯžƳŚĪººſŚººŝƢººǀƤŰţ ŵŹřŶƳŚŤºſřƮŬůŹŵƂƴĩřƹƽŹřźƣźŝƎƿřźƃŚŝƹƝŶƷƽŚƷƱĥ ÑƩƹŶºūŹŵũŹŶºƴƯšŚºŞǀĩźţƽƹŚºůƽŚºƷƶƫƺƫƶŝƵŶƃƶǀƸţ ŹřźºƣƝŹƹŶºƴěřMaster CyclerƵŚĮŤºſŵŹŵƹƵŶºƃƶƟŚƋř ƽƶºƬůźƯƪƯŚºƃƶĩŶƿŵźĭƭŚŬƳřPCRźƿŻƶƯŚƳźŝŚŝƹƶŤƟźĭ ƪƯŚºƃƶºųźģÐÍžĜſƹÖÑƽŚƯŵŹŵƶƤǀƣŵÔƶųźģ Î Ʃƹř ŚºƷźưƿřźěƩŚƈţřƽŚƯŵƶūŹŵÒÒƶƤǀƣŵÎƶūŹŵÖÑƶƬůźƯƶſ ÔƶºūŹŵÔÏŹŵƾƿŚºƸƳƽƶƬůźƯƹƶƤǀƣŵÎƶūŹŵÔÏƶƤǀƣŵÎ řŶŤŝřŹōƾ ſƾ ěžĪƬěƾŤƫƺƯƭŚŬƳřŻřƪŞƣŢƟźĭšŹƺƇƶƤǀƣŵ ŻřŶºƘŝŶƳŶºƃƶºƴǀƸŝŹōƾ ºſƾ ºěƪůřźƯƹŵřƺƯƱřżǀƯĨţĨţ ƽřźºŝƶºƴǀƸŝƎƿřźºƃƹŵřƺƯƱřżǀƯƲƿźţŜſŚƴ ƯƱŵŹƹōŢſŵƶŝ ƵŶºƃƶŤºƃƺƳÑƩƹŶºūŹŵƶºĩ ƾƳřżǀƯŹōƾ ſƾ ě žĪƬě ƾŤƫƺƯ XÍÒŹŵƱƺǀºſŚĪǀƠǀƬǀĜƯōƂƴºĩřƹƭŚưţřŻřžěŶƯōŢſŵƶŝ ŢºƿŚƸƳŹŵŶºƿŵźĭŻŹƺƟƹźŤĪƫřŢƫƹ ÎÍÍ ĥŚŤƫƹŚŝ TBE źƟŚŝ ƭƺƿŶºǀţřĬºƳŹŚºŝŻŹŚºĭōƩĥźºƔƳŵŹƺºƯŶƳŚŝŢƿƹŹŹƺƔƴƯƶŝ ƵŚĮŤºſŵƽƹŹźºŝƺƄºŤƀƃŻřžěƹŶƿŵźĭƽżǀƯōĬƳŹŶƿŚƯƹźŝ ƂƴºĩřƹƶºĩŢºſřźºĩŷƶŝƭŻLJŶƿŵźĭ ƾſŹźŝƂƠƴŝ ƽřŹƹŚƯ žĪƬĜǀŤƫŚºƯƮƷƹžĪƬĜǀƳƺƿšŹƺƇƶŝƮƷƽźŤĩŚŝƶſźƷƽřźŝ ŶƃƭŚŬƳř ŢºǀƇŚºƈŤųřƱŵŹƹōŢºſŵƶŝƽřźººŝŢºǀƇŚºƈŤųřƲºǀºǀƘţ ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ 97 ƱřŹŚƨưƷƹƾƳŚƃƹźƟƽźƠƇžĭźƳ ƽźŤĩŚºŝƶºſƾƇŚƈºŤųřƽŚƷźưƿřźěƶĩƽ ŹƺƏƶŝŶƷŵƾ ƯƱŚƄƳ ÎÓŶºººƃƵŵŚƠŤºººſřžƳŚĪºººſƹŵƽŚƷŶºººǀţƺŘƬĩƺƳšƹŚºººƠţ ƾŘƳƺººſLjĮǀººƃŽƺººŗŹƹřŽƺĩƺĩƺƬǀƟŚŤººſřƽŚººƷƽ źŤĩŚººŝ řŻƺºƴǀūƹźŗōŽŚƳƺƯƹŵƺºſžǀƫŚĪƟŽƺĩƺĩƹźŤƳřƾƬĩŚǀƃźƃř ŚƷƶŤƟŚ ƿ ŵŹřŶƳŚŤſřƽŚƷƶƿƺſMultiplex PCRƹUniplex ƶºĩŶºƴŤƟźĭŹřźƣPCRŵŹƺƯŶƳŶƃƵŵŚƠŤſřƾƠƴƯƩźŤƴĩƱřƺƴƗ žºĪƬě ƾƳƺƿ ƪĪƃƶŝŚƷźưƿřźěƶĩŵřŵƱŚƄƳźưƿřźěŢƠū ÑŚŝ ŢſŚƷźưƿřźěƲƿř ÎÏÖźººưƿřźěŚººŝžĩřźººŤƳōƽŚººƷƶƿƺººſŹŵřŹŻŚººŝŢººƠū ŹŵƭźĭƺƳŚºƳÔÒƩŵŚƘƯƾƯƺƳĥDNAŢƔƬƛƶĩŵƺưƳƆŴƄƯ ƾºſŹźŝÏƪĪºƃŢºſřƵŵźĩŵŚŬƿř ÎÏÒ źưƿřźěŚŝžĩřźŤƳōƹ ŢºƠūÒŚºŝƭƺºƳĥÎÍÑ ŢºƣŹŚºţƞƬŤŴƯ ƽŚƷŢƣŹŚŝ ƾſŹźŝ ƽŚººƷƵŻřŶººƳřŚººŝŶººƳŚŝŹŚººƸģžººĪƬěƾŤƫƺººƯƹžººĪƬěƾƳƺº ƿ ƭƺºƳĥƾºĜĩŵřŶƘţƶĪƴƿřƶŝƶūƺţŚŝƹŢſřŜſŚƴƯŹŚǀƀŝƁƹŹ žǀŤƀºěŚǀƴǀºſźƿ SÎÏźưƿřźěŚŝ ƾƠǀţ LjƳƺưƫŚſ ÎÏÖźưƿřźěŚŝ Î ƩŵŚƘƯƁƹŹŢǀſŚƀůƶŬǀŤƳŹŵŶƃŚŝƾ Ư ƾĜĩ ÏÎÍÓ Ŷūřƹ ƶºſźºƷŹōƾ ºſƾ ěžĪƬěƾŤƫƺƯÐƪĪƃMultiplex PCR ŶºƃŚŝƾ ºƯ ƽźŤĩŚºŝ ÎÍ Śºţ Î ƩŵŚºƘƯƁƹŹŢǀſŚƀºůƲƿřźŝŚƴŝ ƶºĩŢºſřƶºŤƟźĭƭŚŬƳřPCRƾƇŚƈŤųřźưƿřźěŢƠūŪƴěŚŝ ŚººŝƭƺººƿŹƺƯƾººƠǀţLjƳƺưƫŚººſƹžĩřźººŤƳōžǀŤƀººěŚǀƴǀººſźƿ ƶºŝƶºĩŚǀƳƺƯƺƴěLjǀƀŞƬĩżƴƀſŹŚƯŚǀƃřźſƽŶƳƹźƟźŤĩŚŝƹźŤǀſ ŚºƷƶƳƺưƳ ƾſŹźŝƽƶŬ ǀŤƳŵƺƃƾ ƯƵŶƿŵÎƪĪƃŹŵƶĩƱŚƴĤưƷ ƱŵƺŝƾƇŚƈŤųřƽƵŶƴƷŵƱŚƄƳƲƿřƹŶƳŵźĪƳŵŚŬƿřƽŶƳŚŝŪǀƷ ÎÍÕÐƹ ÒÏÍÑÕÖÎÓÑƽŚƷƵŻřŶƳřŚŝŶƳŚŝŹŚƸģžĪƬě ƾŤƫƺƯƹ ęřŹŵƺƳŚºƳƵŚĮŤºſŵŚŝƭƺƳĥŢƔƬƛƽƶŞſŚŰƯŢǀſŚƀůƲǀǀƘţ FźººưƿřźěŚººŝžǀŤƀººěŚǀƴǀº ſźƿTźººưƿřźěŚººŝƾƠǀţLjƳƺưƫŚººſ ŵƺºƃƾ ºƯƵŶºƿŵÑƪĪºƃŹŵƶºĩƱŚºƴĤưƷŶºƃŚŝƾ ƯźŤǀƫƹźĪǀƯ ƪĪºƃƶºŝŚƷźưƿřźěƶĩƵŵřŵƱŚƄƳřŹźưƿřźěŢƠūÑ ŚŝŚƷƶƳƺưƳ ŢǀſŚƀºůƶºĩŶºƷŵƾ ƯƱŚƄƳřŹŜſŚƴƯŶƳŚŝƾƇŚƈŤųřźưƿřźě žĩřźººŤƳōƽŚººƷƶƿƺººſŹŵřŹŻŚººŝŢººƠūÎÓÑÐÔÐÒÏÍÎÍÕÐ ƭƺºƳĥƭźĭƺƳŚƳźƷƖƣřƹŹŵŢſřƭźĭƺƳŚƳÔÒŹŵÎÒÎÍÔźŝřźŝ .ŢººſřƵŵźººĩŵŚººŬƿřÎÏÒźººưƿřźěŚººŝžĩřźººŤƳōƹFźưƿřźěŚººŝ ŵŹřŵřŹƭƺºƳĥŻřƾºĜĩĨƿƽźŤĩŚŝźƷƱƺģƹŢſřƾĜĩÎÍŚţ ƶºĩƽ ŹƺƏƶŝŵřŵƱŚƄƳřŹźƔƳŵŹƺƯƽŚƷƽ źŤĩŚŝŵŹřŶƳŚŤſřƶƿƺſ ŶǀƿŚţřŹƶŬǀŤƳƲƿřżǀƳƾƴƬĩƁŹŚưƃƁƹŹƶŝŢǀſŚƀůƾſŹźŝ ŢººſřƵŵřŵƱŚƄººƳřŹŹŚººƔŤƳřŵŹƺººƯšŚººƘƐƣƭŚººưţŹƺƌººů ƶºƧÎÏÐƪĪºƃƶºŝƶūƺţŚŝPCR Ʊŵƺŝ ƾƇŚƈŤųř ƾſŹźŝ ŵƺưƳ żǀƳřŹŚƷźưƿřźěƹŹōƾ ſƾ ěžĪƬěƾŤƫƺƯƱŵƺŝƾƇŚƈŤųřŪƿŚŤƳ ƢǀƤŰţƲƿřŹŵƵŵŚƠŤſřŵŹƺƯƾƠƴƯƩźŤƴĩƽŚƷƽźŤĩŚŝŢƀǀƫ ÎƩƹŶū ƮƀǀƳŚĭŹřƹźĪǀƯƭŚƳ ƶƿƺſƵŹŚưƃ ƶǀƸţƪŰƯ Staphylococcus areus Shigella sonnei ATCC 25923 ATCC 9290 pasture Iran Esherichia coli ATCC 25922 Enterococcus faecalis ATCC 29212 Pseudomonas aeruginosa ATCC 27853 Citrebacter freundi PTCC 1600 Serratia marcesens PTCC 1111 Klebsiella pneumonia ATCC 7881 ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ Źōƾſ ƾěžĪƬěƾŤƫ ƺƯƽŻřŶƳřƵřŹŚŝƱŚƯżưƷƆǀŴƄţ 98 ƢǀƤŰţƲƿřŹŵƵŶƃƵŵŚƠŤſřƽŚƷźưƿřźěšŚƈŴƄƯÏƩƹŶū ƲǀŘţƹźě ƩƺƈŰƯƵŻřŶƳř ƭƹŻƺƯƹźĩŶǀưſLjěƱĥƵŚĮƿŚū źưƿřźěƵŹŚưƃ invA ÐÔÐbp ƭƹŻƺƯƹźĩ LjƳƺưƫŚſSÎÏ-sÎÐ PXOÎ HindIIIžĩřźŤƳōÎÏÒÎÏÓ ÑÕÖbp hp pA ƭƹŻƺƯƹźĩ ŢƿŚſŶūřƹÎÍÕÐ bp ÎÓÑ bp chr ƭƹŻƺƯƹźĩ ÒÏÍ bp pla SÎÏF: LjƳƺưƫŚſT žĩřźŤƳōÎÏÖ-ÎÐÍ ŚǀƴǀſźƿYer pPstÎ ƢǀƤŰţƲƿřŹŵƵŶƃƵŵŚƠŤſřƽŚƷźưƿřźěžƳŚĪſÐƩƹŶū gta ttg ttg att aat SÎÐR: ata tta cgc acg gag atc cg gaa aca cgt t Salmonella TF: tgt ccg ctg Salmonella TR: atc tca ggc aaa ctc aca agg Antherax ÎÏÒ: tta atg cga Antherax ÎÏÓ: Antherax ÎÏÖ: Antherax ÎÐÍ: Yersinea YerF: gat caa tct gtc atc ttg tct acg g at ttg cga ccg tac ttg aa ccc agg ggg aac gat tgg gaa aca agc tcc aac gat agc tac att tgg act tgc agg cca gta tcg tcc ag c PCRžĪƬěƾŤƫƺƯŶƴƿřźƟŹŵƵŶƃƵŵŚƠŤſřšŚŞǀĩźţƹźƿŵŚƤƯÑƩƹŶū ŹřŶƤƯ ŵřƺƯ ƞƿŵŹ ƩƺƯƺĪǀěÎÔÓ ƾƤƿŹżţźƐƤƯśō Î ƩƺƯƺĪǀěÎ MgclÏmM ƩƺƯƺĪǀěÏÒ ÎÍxźƟŚŝ ƩƺƯƺĪǀěÍÒ dNTPmix (ÎÍÍmM each) ƩƺƯƺĪǀěÎ Rźưƿřźě ƩƺƯƺĪǀěÎ ƩƺƯƺĪǀěÎ ŢǀƳƺƿÏÒ ƩƺƯƺĪǀěÏÒ Fźưƿřźě DNA TaqƮƿżƳř Ɩưū Ï Ð Ñ Ò Ó Ô Õ Ö ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ 99 ƱřŹŚƨưƷƹƾƳŚƃƹźƟƽźƠƇžĭźƳ ÏƽƵŹŚưƃ ƽƶƳŚų ÒÏÍŶƳŚŝƵŻřŶƳřYerźưƿřźěŚŝžǀŤƀěŚǀƴǀſźƿÎƽƵŹŚưƃ ƽƶƳŚų Multiplex PCRƹUniplexŪƿŚŤƳÎƪĪƃ ÎÏÖźưƿřźěŚŝžĩřźŤƳōÑƽƵŹŚưƃ ƽƶƳŚų ÑÕÖŶƳŚŝƵŻřŶƳřTźưƿřźěŚŝLjƳƺưƫŚſÐƽƵŹŚưƃ ƽƶƳŚų ÎÍÕÐŶƳŚŝƵŻřŶƳřÎÏÒźưƿřźěŚŝžĩřźŤƳō YeržǀŤƀěŚǀƴǀſźƿƾƇŚƈŤųřźưƿřźěŢƠūŹŚƸģŚŝŹōƾſ ƾěžĪƬěƾŤƫƺƯ ÓƵŹŚưƃ ƽƶƳŚų ƾƫƺĪƫƺƯƱŻƹÒƽƵŹŚưƃ ƽƶƳŚų ÎÓÑŶƳŚŝƵŻřŶƳř ƾƠƴƯƩźŤƴĩÔƵŹŚưƃƽƶƳŚų ÎÏÖžĩřźŤƳōTLjƳƺưƫŚſÎÏÒžĩřźŤƳō YerźºưƿřźěŚºŝžǀŤƀºěŚǀƴǀºſźƿÎƽƶºƳŚųŢºſřƵŵřŵƱŚƄºƳźºưƿřźěŢºƠūÑŚºŝřŹMultiplex PCRƹUniplexƽƶºŬǀŤƳÏƪĪƃ LjƳƺưƫŚſÑƽƶƳŚų ÎÓÑŶƳŚŝƵŻřŶƳřÎÏÖźưƿřźěŚŝžĩřźŤƳōÐƽƶƳŚų ÎÍÕÐŶƳŚŝƵŻřŶƳřÎÏÒźưƿřźěŚŝžĩřźŤƳōÏƽƶƳŚų ÒÏÍŶƳŚŝƵŻřŶƳř ŻřŢſřšŹŚŞƗƶĩƾƇŚƈŤųřźưƿřźěŢƠūŹŚƸģŚŝŹōƾſ ƾěžĪƬěƾŤƫƺƯ ÓƽƶƳŚųƾƫƺĪƫƺƯƱŻƹ ÒƽƶƳŚų ÐÔÐŶƳŚŝƵŻřŶƳřSÎÏźưƿřźěŚŝ ƾƠƴƯƩźŤƴĩÔƽƶƳŚų ÎÏÒźưƿřźěŚŝžĩřźŤƳōYerźưƿřźěŚŝžǀŤƀěŚǀƴǀſźƿSÎÏźưƿřźěŚŝLjƳƺưƫŚſÎÏÖźưƿřźěŚŝžĩřźŤƳō ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ Źōƾſ ƾěžĪƬěƾŤƫ ƺƯƽŻřŶƳřƵřŹŚŝƱŚƯżưƷƆǀŴƄţ 100 ŚŝžǀſřźŤƳōŽƺƬǀſŚŝŹōƾſ ƾěžĪƬěƾŤƫƺƯ ÎƽƶƳŚų ŢſřƵŵřŵƱŚƄƳřŹƾƇŚƈŤųřźưƿřźěŢƠūŪƴěŚŝMultiplexPCRŪƿŚŤƳÐƪĪƃ źưƿřźěŚŝžǀŤƀěŚǀƴǀſźƿÑÕÖŶƳŚŝƽƵŻřŶƳř TźưƿřźěŚŝLjƳƺưƫŚſÐÔÐŶƳŚŝƽƵŻřŶƳř SÎÏźưƿřźěŚŝLjƳƺưƫŚſÎÓÑŶƳŚŝƽƵŻřŶƳř ÎÏÖźưƿřźě ƾƠƴƯƩźŤƴĩÐƽƶƳŚųƾƫƺĪƫƺƯƱŻƹ ÏƽƶƳŚų ÎÍÕÐŶƳŚŝƽƵŻřŶƳř ÎÏÒźưƿřźěŚŝžǀſřźŤƳōŽƺƬǀſŚŝÒÏÍŶƳŚŝƽƵŻřŶƳř Yer śÑƪĪƃƞƫřÑƪĪƃ ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ 101 ƱřŹŚƨưƷƹƾƳŚƃƹźƟƽźƠƇžĭźƳ ŵÑƪĪƃũÑƪĪƃ ƵÑƪĪƃ ŢƠūŚŝDNAƩŚƿźſŢƣŹƞƫřÑƪĪƃÑÕÖŶƳŚŝƽƵŻřŶƳř ƾƠǀţLjƳƺưƫŚſTźưƿřźěŢƠūŚŝDNAƩŚƿźſŢƣŹƶŝƍƺŝźƯŪƿŚŤƳÑƪĪƃ ŢƣŹũÑƪĪƃÎÓÑŶƳŚŝƽƵŻřŶƳř žĩřźŤƳōÎÏÖźưƿřźěŢƠūŚŝDNAƩŚƿźſŢƣŹśÑƪĪƃÐÔÐŶƳŚŝƽƵŻřŶƳř SÎÏÎÐźưƿřźě žǀŤƀěŚǀƴǀſźƿYerźưƿřźěŢƠūŚŝDNAƩŚƿźſŢƣŹƹŵ ÑƪĪƃÎÍÕÐŶƳŚŝƽƵŻřŶƳř žĩřźŤƳōÎÏÒźưƿřźěŢƠūŚŝDNAƩŚƿźſ ÎÍ-ŢƣŹŚŝDNAÏƽƶƳŚų ƆƫŚųƭƺƳĥƶƳƺưƳÎƽƶƳŚų śƹƞƫÎÑƪĪƃsÎÏƹTźưƿřźěŚŝLjƳƺưƫŚſƵÑƪĪƃÒÏÍŶƳŚŝƽƵŻřŶƳř ÎÏÒźưƿřźěŚŝžĩřźŤƳōƾƫƺĪƫƺƯƱŻƹÓƽƶƳŚų ÎÍ-ÑŢƣŹŚŝDNA.ÒƽƶƳŚų ÎÍ-ÐŢƣŹŚŝDNA ÑƶƳŚųÎÍ-ÏŢƣŹŚŝDNAÐƽƶƳŚų Î ƽƶƳŚų ƵÑƪĪƃÒÏÍŶƳŚŝƵŻřŶƳřYerźưƿřźěŚŝžǀŤƀěŚǀƴǀſźƿƹũ ÑƪĪƃÎÓÑŶƳŚŝƵŻřŶƳřÎÏÖŵ ÑƪĪƃÎÍÕÐŶƳŚŝƵŻřŶƳř ÎÍ-ÐŢƣŹŚŝDNAÒƽƶƳŚų ÎÍ-ÏŢƣŹŚŝDNAÑƽƶƳŚų ÎÍ-ÎŢƣŹŚŝDNAÐƽƶƳŚų ƾƫƺĪƫƺƯƱŻƹÏƽƶƳŚų ƆƫŚųDNA Î ÎÍ-ÑŢƣŹŚŝDNAÓƽƶƳŚų HindIIIƮƿżƳōƎſƺţPCRƂƴĩřƹÎÍÕÐbpƩƺƈŰƯƁźŝÒƪĪƃ ƽŚƷƵŻřŶƳřŚŝƶƘƐƣƹŵHindIII ƵŶƴƷŵƁźŝźŧLJřŵƹŶŰƯƮƿżƳō ƾŤºſŹŵƽƵŶºƴƴĩŶºǀƿŚţƶºĩŵƺƃƾƯƪƇŚůÐÕÓbpƹÓÖÓbp źưƿřźěŚŝžĩřźŤƳōMultiplex PCRƂƴĩřƹƩƺƈŰƯŶǀƿŚţ ƎºſƺţÎÍÕÐ bpƩƺƈŰƯƾưƿżƳōƁźŝŻřƪƇŚůŪƿŚŤƳ ÎÏÒ ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ Źōƾſ ƾěžĪƬěƾŤƫ ƺƯƽŻřŶƳřƵřŹŚŝƱŚƯżưƷƆǀŴƄţ šƹŚºƠŤƯƽŚºƷƵŵřƺƳŚºųŻřƞƬŤŴƯƶƳƺĭƶſźǀųřƢǀƤŰţƁƹŹ 102 ƽƶƀºƿŚƤƯÒƪĪºƃŢſŚƷźưƿřźěƎſƺţƵŶƃŵŚŬƿřƩƺƈŰƯ ŹƺºƏƶºŝšƹŚºƠŤƯƾƴǀƫŚºŝƹ ƽźƷŚºƓ ƽŚºƷšƹŚºƠţŚŝŚƷƽ źŤĩŚŝ invAƱĥƹÎÏƽŚººººƷźưƿřźěPCRƩƺƈººººŰƯƝŵřźººººţ ƽŚºƷźưƿřźě ƾºƠǀţLjƳƺưƫŚſ ƾƫƺĪƫƺƯƆǀŴƄţŢƸūƲǀƤƤŰƯ ƽŚƷŶºǀţƺŘƬĩƺƳŶºƃŚŝƾºƯƖºūźƯƱĥžƳŚĪºſƾƳŚŤŰţƽƶŤƃŹ ŶººƳŵƺưƳƾººſŹźŝƹƾººůřźƏřŹd,flic-a,viaB,prt,spvC ÏƽƶºƳŚųƾƫƺºĪƫƺƯƱŻƹÎƽƶºƳŚųŢºſřƵŶºƿŵźĭƆŴƄƯ ÎÔŢƟźĭŹřźƣ ƾƿŚſŚƴƃŵŹƺƯƂƿŚƯŻōƶƫƺƫĨƿ ŹŵƱŚƯżưƷ tyv,flic-ƵŶºƃƶŤųŚƴºƃ ƽŚºƷƱĥƞºƿŵŹŽŚſřźŝ ƾƠƬŤŴƯ ƶºĩŢºſřƲºƿřźºƋŚůƢºǀƤŰţŚºŝƢºǀƤŰţƲºƿřšƹŚƠţÎÕ źºŝƱŚºƯŻƶºĩŢºƟźĭŹřźºƣƾºůřźƏŵŹƺƯƾƠƬŤŴƯƽŚƷźưƿřźě ŚºƸƴţ ƵŵŚƠŤºſřŵŹƺºƯ ƽŚºƷƱĥƹŢºſřźţŹřƺºƃŵ ŹŚĩƹŶƴŤƀƷ ƾƴǀƫŚºŝƽŚºƷƶºƳƺưƳŻřŶºƿŚŝŚºưŤůƶĩŶƳŵƺŝžƳLJƹźƿƹƽŚƷƱĥ ƹ PCR ƩƺƈºŰƯŻřƪºƇŚůžƳŚĪſƾƳŚƣƺƟƶŤƃźƳĥĨƳŚŝŻř ÏÐÐƹÏÐÏÎÏÑƵŚººĮƿŚūŹŵźŤŤººƃŹŵƝƹźººůŚººŝšƹŚººƠŤƯ ƮƌºƷƩƺƈºŰƯÐƽƶƳŚųÎÍÕÐbp PCR ƂƴĩřƹƩƺƈŰƯ ƵŶºƃƮƌºƷƩƺƈºŰƯÑƽƶƳŚųHindIII ƮƿżƳōƎſƺţƵŶƃ HindIIIƮƿżƳōƎſƺţ ƽŹŚưǀŝƆǀŴƄţƽřźŝžƳLJƹźƿƹźǀƛƮƷƹžƳLJƹźƿƹƽŚƷƱĥ ŦŰŝ ƶºĩŶºƃƵŵŚƠŤºſřŵŹřŶƳŚŤºſř ƽŚºƷƶƳƺưƳŻř ƾſŹźŝƲƿřŹŵ ƁƹŹŚººŝƹviaB,fliC,tyv(rfbE), prt(rfbS)ƽŚººƷƱĥ ƮǀƤŤƀƯƽƶƳƺưƳƲ ǀƴĤưƷƹƽźŤĩŚŝƶſƲƿřƱLJƹźƿƹƪƯŚƗƭƺƳĥ ƲǀºƗŹŵƹƵŵƺºŝŵŚƿŻƁƹŹƲƿř ƽŚƷźưƿřźěŵřŶƘţÎÖŢſř ŵƺūƹƾĪǀƴǀƬĩƽƶƳƺưƳŹŵŢſřƲĪưƯƶĩƽźĮƿŵ ƽŚƷƽ źŤĩŚŝ ƪºƯŚƗƆǀŴƄºţŢºƸūżºǀƳ ƾºƠƬŤŴƯŹŚǀƀŝ ƽŚƷƁƹŹ Ţſř ŚºŝƶºƘƐƣŹŚƸģĨǀĪƠţƱŚĪƯřƶĩŢſř ƽƺŰƳƶŝƵŶƃśŚŴŤƳř ŶºƴƯŻŚǀƳƆǀŴƄºţŢºƸūŵƺºūƺƯŵŹřŶƳŚŤſř ƽŚƷƁƹŹ Ţſř ŶƇŹŵÎÒŚţ Î ŢƔƬƛŚŝŻŹƺƟźŤĪƫřƩĥŻŹŚĭō ƽŵŚƗƮŤƀǀſŚŝ ƾƫƺºĪƫƺƯƞƬŤŴƯƽŚƷƁƹŹŻřÏÍÏÏŶƃŚŝƾ ƯƽźŤĩŚŝƲƿř Ɩƿźºſ PCRƽŚºƷƶųźģƭŚŬƳřƂƴĩřƹƍƺƬŴƯ ƽƶǀƸţƭƺƳĥ ƽřźººŝƁƹŹƲƿźººŤƸŝŚººƯřŢººſřƵŶººƃƵŵŚƠŤººſřŚººƷŽƺƬ ǀº ſŚŝ ƽŚºƷƁƹŹ ŹŵŵŹřŵŵƺºūƹƶƤǀƣŵÖÍŻřźŤưĩŹŵUVƵŚĮŤſŵ ŹřźºƣƵŵŚƠŤºſřŵŹƺºƯƢºǀƤŰţƲƿřŹŵƶĩŢſřŹōƾ ſƾ ěĨǀƴĪţ ŶºƃŚŝƾ ºƯƽźŤĩŚºŝƲƿřƖƿźſƆǀŴƄţŢƸūƭŻLJƾƿřŹŚĩŶƣŚƟ ƽřźººŝƽŹŚººưǀŝƲº ƿřƭŚººĮƴƷŵƹŻƹƖººƣƺƯƶººŝƆǀŴƄººţƽřźººŝ ƪººƯŚƗƆǀŴƄººţŢººƸūPCRƁƹŹŻřƶŝŚƄººƯƢºǀƤŰţĨºƿ ŹřźºƣƵŵŚƠŤºſřŵŹƺƯ ƾĪƿĥƺƫƺǀŝ ƽŚƸŞưŝŹŵƶĩřƺƷŹŵžĩřźŤƳō ƶºĩƽźŤĩŚºŝƕƺºƳĨºƿŚºƸƴţƶĩŢſřƵŶƃƵŵŚƠŤſřŚƷLjƳƺưƫŚſ ŻřƱřƺºţƾ ºƯƮºƷźƋŚůƂƿŚƯŻōŪƿŚŤƳŹŵŚƯřŶƳƺƃƶǀƸţƵŵƺƫō źºŝ ƾºƴŤŞƯźºưƿřźěŢºƠūŪƴºěŚŝ ƾſŹźŝĨƿŹŵ ŵźĩƵŵŚƠŤſř ƆǀŴƄºţƶºŝŹŵŚºƣƾƇŚƈŤųřŹƺƏƶŝƵŶƃśŚŴŤƳřƽŚƷźưƿřźě ƵŵƺºŝLjƳƺưƫŚºſƪƯŚƗƆǀŴƄţƶŝŹŵŚƣƾŝƺųƶŝžĪƬěƾŤƫƺƯ ŚºƿƹƵŵřƺƳŚºųƮƷƽŚƷƽ źŤĩŚŝźƿŚſŚŝƾŴſŚěƶƳƺĭĢ ǀƷƹƵŵƺŝ ƩƹřŶºŤƯŢƗźºſŚŝ ƽŵŚƗ PCR ƵŶƃƶŤƟźĭŹŚĩƶŝƁƹŹƩŚů ƽŚºƷźưƿřźěšŚºƘƐƣƽƵŻřŶºƳřƹŶºƳřƵŵźºĪƳŵŚŬƿřŶƃŚŝƶŤƃřŵ ƵŶºƃƵŵŚƠŤºſř ƾƐǀŰƯƹ ƾƯřŵ ƾĪǀƴǀƬĩ ƽŚƷƶƳƺưƳ ŹŵžĩřźŤƳō ŢƫƺƸºſƶºŝŻŚŝŢƠūÎÍÕÐÑÕÖÒÏÍÐÔÐÎÓÑƽŚƷƵŻřŶƳř ƖƿźſƆǀŴƄţŢƸūƭŻLJ ƾƿřŹŚĩŶƣŚƟƶĩŢſř ƾƳLJƺƏƱŚƯŻ ƖƿźºſƆǀŴƄºţƱŚºĪƯřƁƹŹƲºƿřŻřƵŵŚƠŤºſřŚŝŵŹřŵŵƺūƹ źƿŚºſŻřƱōĨºǀĪƠţƹžĩřźºŤƳōƪƯŚƗ ƽźŤĩŚŝƆǀŴƄţ ƽřźŝ ŹŵƶºŬǀŤƳƾºſŹźŝƹŶƿŚƯƹźŝƭƺƿŶǀţřƵŵŚƯƽƹŚůŻŹƺƟƹźŤĪƫř ŻřƵŵŚƠŤºſř ƾƫƺºĪƫƺƯƁƹŹƱŚºưƷžĩřźºŤƳōƪºƯŚƗƆǀŴƄţ ƶºĩŢºſřƾƳLJƺºƏƱŚƯŻŶƴƯŻŚǀƳŚƷLjƳƺưƫŚſƆǀŴƄţƩƹřŶŤƯ ŢºſřƾţŚºǀůŹŚǀƀŝƶĩƱŚƯŻŹƺŤĩŚƟŻřƵŵŚƠŤſř ŢƬƗƶŝ ŢƟźĭ ŹŵÖŵƺºƃƾ ƯƭŚŬƳřƖƿźſŹŚǀƀŝƆǀŴƄţƁƹŹƲƿřŹŵŚƯř ŹƺĜºſřƂºŴěƅƺƈºųƶŝƽŹŚưǀŝƱŶƃƾƯŶǀěřŻřƽźǀĭƺƬū źƿŚſƲǀƴĤưƷƹŚƷƶſŚƿźŤĩŚŝƹźŤƳřźƿŚſŻřƱōĨǀĪƠţƹƶŞƈů šŚºƘƫŚƐƯŹŵÎÖÖÐƩŚſŹŵƭřƺƃƹũŚŞƴǀƷÏÐÏÕŵźǀĭƾ Ư ŹŵŚºƯř ŢºƟźĭŹřźºƣ ƾƿŚſŚƴƃŵŹƺƯ ŢſřƶŞƈůƪƯŚƗƱŚưƷ ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ 103 ƱřŹŚƨưƷƹƾƳŚƃƹźƟƽźƠƇžĭźƳ ƁƹŹŻřƾºƬưƗƽźǀĭŹŚºĩƶºŝŹŵƾºſŚſřƪºƯŚƗŵźºĩƵŵŚƠŤſř ŢºŰţŹŵžǀŤƀºěŚǀƴǀºſźƿƽźŤĩŚºŝƭƺºƳĥƾºſŹźŝ ƽřźŝŵƺų ŢºƸūƲǀºƤƤŰƯƶºĩŢſřƽŹŚǀƀŝƽŚƷƵŚĮ ƿŚū ƾſŹźŝŶƴƯŻŚǀƳ šŚƤǀƤŰţŪƿŚŤƳƹŶƳŵźĩƵŵŚƠŤſř ƽźŤĩŚŝ ƾƘƐƣ ƾƿŚſŚƴƃ ŢƸū źºưƿřźě ƾºůřźƏŢſř ƾƇŚƈŤųř ƽŚƷźưƿřźě ƾƿŚſŚƴƃ PCR ŻřƾºƠƬŤŴƯƽŚºƷƂŴŝŻřŜſŚƴƯ ƽŚƷśƹźěƹźưƿřźě ƾůřźƏ ŶƳŵźĩƵŵŚƠŤſřŵƺūƺƯƽŚƷƾ ƫřƺţ ƽźǀĭƶŬǀŤƳ ŹōƾºſƾºěžºĪƬěƾŤƫƺƯƁƹŹƢǀƤŰţƲƿřŪƿŚŤƳŽŚſřźŝ LjƳƺưƫŚſƽźŤĩŚŝƶſƱŚƯżưƷƹƖƿźſƆǀŴƄţŢƸūŶƳřƺţƾƯ źƿŚºſŻřžǀŤƀºěŚǀƴǀºſźƿƹžǀſřźŤƳōŽƺƬǀſŚŝƭƺƿŹƺƯƾƠǀţ ŹřźºƣƵŵŚƠŤºſřŵŹƺºƯŚºƷƱōƵŵřƺƳŚºųƮºƷŚƿƶŝŚƄƯƽŚƷƽźŤĩŚŝ ƽŚƷŭLjſƱřƺƴƗƶŝƪƯŚƗƶſƲƿřŻřƶĪƴƿřƶŝƶūƺţŚŝƹŶƳźǀĭ ŶºƳřƺţƾºƯƶºƘƫŚƐƯƲºƿřŵźºŝƭŚƳƱřƺţƾƯħŚƳźƐųƹƾŝƹźĪǀƯ ƽřźººŝƶººƴƿżƷƮººĩƹƢººǀƣŵƖƿźººſƆǀŴƄººţƽřźººŝƽŹřżººŝř ƹƾŤƀƿŹƹźţƺǀŝšŚǀƬưƗŶƴƳŚƯƽŵŹřƺƯŹŵŚƷƽźŤĩŚŝƲƿřƾŝŚƿŵŹ ŹŚǀƀºŝƁƹŹŢǀƇŚƈºŤųřŶƿŚưƳƮƷřźƟƾŝƹźĪǀƯƽŚƷĬƴūŚƿ ƲǀºŝŹŵřŹƽźŤĩŚºŝƕƺºƳĨºƿŢſřŹŵŚƣƶĩƽŹƺƏ ƶŝŢſřLJŚŝ ƁƹŹƲºƿřƭŚºŬƳřŶºƷŵƆǀŴƄţšƹŚƠŤƯƽźŤĩŚŝƕƺƳƱřŹřżƷ šŹƺºƇƱŚºƯżưƷŹƺºƏƶŝƶĪƴƿřƶŝƶūƺţŚŝƹŢſřƵŵŚſŹŚǀƀŝ źºƷƆǀŴƄºţŢºƸūŵŶƘŤƯƽŚƷ PCRƭŚŬƳřƶŝŻŚǀƳŵźǀĭƾ Ư ƩŚºſƵŵśŹŚºŬţƶºŝƶºūƺţŚŝŶƃŚŝƾ ưƳƶƳŚĭřŶūŹƺƏƶŝƪƯŚƗ ƵŶºƃƭŚŬƳřƽŚƷƽ ŻŚſƶƴǀƸŝƹżĩźƯƲƿřŹŵƵŶƃŜƀĩƶŤƃŸĭ ŢººƸūƾº Şǀĩźţ PCRƭŚººŬƳřƪººƯřƺƗĨººţĨººţŵŹƺººƯŹŵ ƩƺºƸŬƯƽŚºƷƶƳƺưƳŹŵƪƯŚƗŶƴģƱŚƯżưƷƹƖƿźſƆǀŴƄţ ŹŵƱřƺºţƾ ƯƁƹŹƲƿřŻřŢſřƁƹŹƲƿřŶƴưƃŻŹř ƾƿŚƿřżƯŻř ŚººƿƹƾƳƺººƠƗƪººƯřƺƗƾƫƺººĪƫƺƯƆǀŴƄººţƽŚººƷƵŚĮƄ ººƿŚƯŻō ƅŚųƎƿřźƃƹŚƷƱřźŰŝŢƸūƵŶƃƶǀƸţŹŚǀſ ƽŚƷƵŚĮƄ ƿŚƯŻō ƆǀŴƄºţƖºūźƯƽŚºƷƵŚĮƄƿŚƯŻōŹŵƲǀƴĤưƷƹŵƺưƳƵŵŚƠŤſř ŹŵƪºƯřƺƗƱŚºƯżưƷƆǀŴƄţƶŝƾĭŹżŝĨưĩŶƳřƺţƾ ƯƪƯřƺƗ ƲǀºƴģƱƺºƴĩŚºţƱŚƸūŹŵƶĪƴƿřƶŝƶūƺţŚŝŶƿŚưƳƂƴĩřƹĨƿ ƵŶºƃźºĩŷƪºƯŚƗƶºſƖƿźſƹƱŚƯżưƷƆǀŴƄţ ƽřźŝ ƾƃƹŹ plaƱĥƽŚºƷźưƿřźěŻřŚĪƿźƯōƹŚƤƿźƟōŚǀſōƞƬŤŴƯƽŚƷƶƳƺĭ ƝřŶƷřƶŝƪǀƳŢƸū Źŵ PCR ƂƴĩřƹƭŚŬƳřƶĩŵřŵƱŚƄƳŚƷƱō ƪºŝŚƣƽĥƺƫƺǀƯŶºǀěřƽŚºƷƾ ſŹźŝƹƽŹŚưǀŝƾƫźŤƴĩƹƾƈǀŴƄţ ƁƹŹƶºĩŢºſřƲƿřŹŵźƋŚůƂƿŚƯŻōŚŝšƹŚƠţŢſřŵŚƴŤſř ŚºƯƢºǀƤŰţŚºƯřŢſřŹōƾ ſƾ ěžĪƬěƾƳƺƿ ŚƸƴţƵŵŚƠŤſřŵŹƺƯ ŦººƗŚŝƶººĩƵŶººƃƵŵŚƠŤººſřŹōƾ ººſƾ ººěžººĪƬěƾŤƫƺººƯƁƹŹ ƆǀŴƄººţƹƵŵŚƠŤººſřŵŹƺººƯŵřƺººƯƹƱŚººƯŻŹŵƾƿƺººūƶƟźººƇ ÏÖŢºſřƵŶºƃƮºƷŚŝƱŚƯżưƷŚƷƽ źŤĩŚŝƞƬŤŴƯ ƽŚƷƶƳƺĭ ƽźŤĩŚŝ ƾƿŚſŚƴƃ ƽřźŝ ÎÖÖÖ ƩŚſŹŵżǀƳƂƳřŹŚĪưƷƹźƫŚŤƬĮƳř ƱřƺººǀůƶººŝŮǀººƤƬţƁƹŹŚººŝřŹPCRƁƹŹƱƺƗŚººƏƪººƯŚƗ ƶºĩŵřŵƱŚƄºƳŚƷƱōšŚƤǀƤŰţŪƿŚŤƳŶƳŵźĩƶƀƿŚƤƯƾƷŚĮƄƿŚƯŻō ŚººƸƴţƶººƳPCRƁƹŹŚººŝƱƺƗŚººƏƪººƯŚƗƽźŤĩŚººŝƾƿŚººſŚƴƃ šŚºƳřƺǀůŻřƾºųźŝŢºƯƹŚƤƯƪǀƫŵƶŝřŹśŷŚĩƾƠƴƯšLjĪƄƯ ƾºƃƹŹƶºĪƬŝŶºƴĩƾ ºưƳŵŚŬƿřžǀŤƀěŚǀƴǀſźƿ ƶŝ ƾƷŚĮƄƿŚƯŻō ŶºƃŚŝƾ ºƯƱŚºƯŻƹšŚºƳŚĪƯřƽƶºƴƿżƷźƔƳŻřƶƟźƇƶŝƱƹźƤƯ ƽŚºƷƁƹŹƶºŝŢŞƀºƳƁƹŹƲºƿřƶºĩŶºƴŤƟźĭƶºŬǀŤƳƲƿřźŝŚƴŝ ƶºŝŮǀºƤƬţƹ ƽĥƺƫƺƿźŤĩŚºŝŢƄºĩƱƺºĤưƷ ƾƈǀŴƄţƩƺưƘƯ ŶƳřƺţƾ ƯƹŢſřźţƢ ǀƣŵƹźţŽŚƀůŹŚǀƀŝƾƷŚĮƄƿŚƯŻōƱřƺǀů ŢººŞƣřźƯƹƩźººŤƴĩƆǀŴƄººţŢººƸūŹŵŜººſŚƴƯƁƹŹĨº ƿ ƶŝŢŞƀƳžĪƬěƾ ŤƫƺƯƁƹŹÐÍŶƿōŹŚưƃƶŝƱƺƗŚƏ ƽŹŚưǀŝ ƶĪƬŝŶƃŚŝƾ ƯźţƖ ƿźſƹźţƶƴ ƿżƷƮĩŚƸƴţƶƳžĪƬěƾƳƺƿƁƹŹ ŹŵŵźºĩŶºƷřƺųƝŸºůƪƯŚĩŹƺƏƶŝżǀƳřŹśŷŚĩƾƠƴƯŵŹřƺƯ ƾºƠǀţLjƳƺưƫŚſhpƹinvAƽŚƷƱĥŽŚſřźŝƶĩƢǀƤŰţƲƿř ƾƯŚºưţŶºƃƭŚŬƳřžǀŤƀěŚǀƴǀſźƿplaƹžĩřźŤƳōchrƹpA ƾƿŚºſŚƴƃŵŹƺºƯŵƺºųƾƇŚƈŤųřźưƿřźěƶŝƶūƺţŚŝŚƷƱĥƲƿř ƽŚºƷƶºƇźƗŹŵƮǀºƔƗ ƽŚºƷŢƟźƄ ºǀěƶŝƶūƺţŚŝŢƟźĭŹřźƣ Ʊřƺºţƾ ºƯ Źōƾ ſƾ ěžĪƬě ƾŤƫƺƯƪŨƯŚƷƁƹŹƲƿřŻř ƾƫƺĪƫƺƯ ƲºƿřƆǀŴƄţƶƬưūŻřƾƳƺƠƗƞƬŤŴƯƪƯřƺƗƆǀŴƄţŢƸū žǀŤƀºěŚǀƴǀºſźƿƭƺºƿŹƺƯƾºƠǀţLjƳƺưƫŚſžĩřźŤƳōƪƯŚƗƶſ ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ Źōƾſ ƾěžĪƬěƾŤƫ ƺƯƽŻřŶƳřƵřŹŚŝƱŚƯżưƷƆǀŴƄţ 104 šŚºƤǀƤŰţżºĩźƯŻřƭźºŤŰƯƱřŹŚĪưƷƹŶǀţŚſřƹŪƗĽřƶǀƤŝ ŚºŝƹŵźºƟƶŝźƈŰƴƯƱřƺţƾ ƯřŹƢǀƤŰţƲƿř ŢſřƶŤƟŚǀƳƶƘſƺţ ƢºǀƤŰţƲºƿřƭŚºŬƳřŚºƷƱōƶŞƿŚºƃƾ ŝ ƽŚƷšŶƗŚƀƯ ƹ ƽŹŚĪưƷ ŶƃŚŝƾ ƯƾƫƺĪƫƺƯƆǀŴƄţ ƱƹŶººŝƶººĩƾº ƯŻŹŜººƏƽƵŶĪƄººƷƹĦěƹƾƫƺººĪƫƺƯƽĥƺººƫƺǀŝ ƽƺŬƄºƳřŵƶºƯŚƳƱŚºƿŚěƪºƇŚůƶºƫŚƤƯƲºƿřŵƺºŞƳźƿŸºěƱŚĪƯř ŶƃŚŝƾ ƯŶƃŹřƾſŚƴƃŹŚĩ ƽŚºƷŢ ºǀĩŢųŚºſŢºƸūƶƘſƺţƪŝŚƣŚƘƐƣƹŢƀƳřŵƁŻŹř źĪƄţƹźƿŶƤţ ƾĪºƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƭźŤŰƯŢƳƹŚƘƯŻřźĪƄţŚŝ References phospholipase D activity required for virulence in 1- Crump JA, Luby SP, Mintz ED. The global mice. Int J Med Microbiol. 2000; 290: 483-487. burden of thyphoid fever. Bull World Health Org. 8- Brubaker RR. Factors promoting acute and 2004; 82: 346-53. chronic 2- Purver R. Chemical and biological terrorism. Microbiol Rev. 1991; 4: 309-324. The threat according to the open literature. 1995. 9- Ivanhoff B. Typhoid fever global situation and www.csis-scrs.gc.ca/eng/misc docs/pur V_e.html. WHO recommendations. Southeast Asian J Trop 3- Okinaka R, Cloud K, Hampton O, Hoffmaster Med Public Health. 1995; 26: 1-6. A. Sequence, Assembly and analysis of Pxo1 10- Reeves MW, Evins GM, Heiba AA, Plikaytis and Pxo2. J Applied Microbiol. 1999; 87: BD, Former JJ. Clonal nature of Salmonella typhi 261-62. and its genetic relatedness to other Salmonella as 4- Franz DR, Jahrling PB, Fried lander AM, Mc shown by multilocus enzyme electrophoresis, and Clain DJ, Hoover DL, Bryne WR, et al. Clinical proposal of Salmonella bongori Comb nov. J Clin recognition and management of patients exposed Microbiol. 1989; 27: 313-330. to biological warfar agents. JAMA. 1997; 278: 11- 399-41. Cosivio.Guidelines for the surveillance and 5- Inglesby TV, Dennis DT, Henderson DA, control of anthrax in human and animals. WHO Bartlett JG, Ascher MS, Eitzen E, et al. Plague as Publication. 1998. a biological weapon medical and public health 12- Leal NC, Almeida AM. Diagnosis of plague management. JAMA. 2000; 283: 2281-2290. and identification of virulence markers in yersinia 6- Butler T. Plague and other Yersinia infections. pestis by multiplex PCR. Rev Inst Med Trop Sao NewYork Plenum Medical Book Company. 1991; Paulo. 1999; 41: 339-342. 112. 13- Hu Wung, Yujun Cui, Zuyu Wung. Xiaoyi 7- Hinnebusch J,Cherepanov P, Rudoplh A, et al. Wang, Zhaobiao Guo, Yanfeng Yan, et al. A Dog – Murine Associated primary pneumonic plague in Qinghai toxin of yersinia pestis shows disease Turnbull caused PC B, by yersinia. Boehm R, Clin and ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ 105 ƱřŹŚƨưƷƹƾƳŚƃƹźƟƽźƠƇžĭźƳ province, China. Clin Infect Dis. 2011; 52: 185- protective antigen in patients with bioterrorism 190. related cutaneous or inhalation anthrax. J Infect 14- Turnbull PC. Bacillus antheracis but not Dis. 2004; 190: 1228-1236. always antherax. J Appl Bacteriol. 1992; 72: 21-8. 22- Biagini RE, Sammons DL, Smith JP, Page EH, 15- Joseph Sambrook and David W. Russel, Snawder Molecular Cloning: A Laboratory Manual, Cold Determination of Serum IgG antibodies to bacillus Spring Harbor Laboratory Press. 2000. anthracis protective antigen in environmental 16- Altschul SF, Madden TL, Schaffer AA, et al. sampling workers using a fluorescent covalent Gapped BLAST and PSI- BLAST: A new microsphere immuno assay. Occup Environ Med. generation of protein database search programs. 2004; 61: 703-708. Nucleic Acids Res. 1997; 25: 3389-3402. 23- Welkos SL, Lowe JR, Eden MC Cutchan F. 17- Aabo S, Ramussen DF, Rossen L, Sorensen Sequence and analysis of the DNA encoding PD, Olsen JE. Salmonella identification by protective antigen of polymerase chain reaction. Mol Cell Probes. 1993; 1988; 69: 87-300. 7: 171-178. 24- Ramisse V, Patra G, Garrigue H. Identification 18- Hirose K, Itoh KI, Nakajima H, Kurazono T, and characterization of Bacillus antheracis by Yamaguchi M, Moriya K, et al. Selective multiplex PCR analysis of sequences on plasmids amplification of tyv(rfbE), prt(rfbS),via B, and fliC Pxo1 and Pxo2 and chromosomal DNA. FEMS genes by Multiplex PCR for identifications of Microbiol Let. 1996; 9-16. Salmonella enterica Serovars typhi and para typhi 25- Beyer W, Glockner P, Otto J, Bohm H. A A. J Clin Microbiol. 2002; 40: 633-636. nested PCR method for the detection of Bacillus 19- Herrera Leon S, Mc Quiston JR,Usera MA, anthracis in environmental samples collected from Fields PI, Garaizar I, Echeita MA. Multiplex PCR former tannery sites. FEMS Microbiol Lett. 1995; for distinguishing the most common phase-1 128: 113-8. flagellar antigens of salmonella. J Clin Microbiol. 26- Handerson I, Duggleby CJ, Tunder bull PC. 2004; 42: 2581-86. Differntation of Bacillus anthracis from other 20- Harrison LH, Ezzel JW, Abshire TG. Bacillus cereus group bacteria with the PCR. Int J Evaluation of serologic tests for diagnosis antherax Bacteriol. 1994; 44: 99-105. after an outbreak of cutaneous antherax in 27- Beyer W, Pocivalsek S, Bohm R. Polymerase Paraguay. J Infect Dis.1989; 160: 706-10. chain reaction-ELISA to detect Bacillus anthracis 21- Conrad P. Quim, Peter M. Dull, Vera from soil samples limitation of present published Semenova, Han Le, Shan Crotty, Thomas H, primers. J Appl Microbiol. 1999; 87: 229-360. et al. Immne Responses to bacillus anthracis 28- Jackson PJ, Hugh Jones ME, Adair DM. PCR JE, Striley CAF, Mackenzie BA. Bacillus anthracis .Gene ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ Źōƾſ ƾěžĪƬěƾŤƫ ƺƯƽŻřŶƳřƵřŹŚŝƱŚƯżưƷƆǀŴƄţ 106 analysis of tissue samples from the 1979 Sverd reaction to detect yersinia pestis in fleas. J Clin lovsk anthrax victims: The presence of multiple Microbiol. 1993; 31: 1511-1514. Bacillus antheracis strains in different victims. 30- Engelthaler DM, Gage KL, Montenieri JA, Proc NatI Acad Sci USA. 1998; 95: 1224-9. Chu M, Carter LG.PCR detection of yersinia pestis 29- Hinne busch J, Schwan TG. New method for in fleas comparison with mouse inculation. J Clin plague Microbiol. 1999: 1980-1984. surveillance using polymerase chain ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ 107 ƱřŹŚƨưƷƹƾƳŚƃƹźƟƽźƠƇžĭźƳ Simultaneous Detection of Salmonella typhi, Bacillus antheracis, and Yersinea pestis by Multiplex PCR Safari Foroshani N1, Karami A2, Pourali F2, Aeidi A1, Fatemeh tavana BS2 1 Islamic Azad University, Science and Research, Tehran, Iran. 2 Research Center of Molecular Biology, Baqiyatallah University of Medical Sciences,Tehran, Iran Corresponding Author: Karami A, Research Center of Molecular Biology, Baqiyatallah University of Medical Sciences, Tehran, Iran. Email: [email protected]. Received: 3 Oct 2012 Accepted: 17 Feb 2013 Background and Objectives: Salmonella typhi, Bacillus anthracis, and Yersinia pestis are potent human pathogenic bacteria that cause typhoid fever, anthrax, and plague,respectively. The classic microbiological methods for detection and identification of these agents are laborious and time consuming.Therefore, developing an accurate method for rapid detection of these potent human pathogens is important, especially due to their potential use as Bioterrorism agents. We have designed a new rapid molecular detection method by using multiplex PCR in a single reaction. Materials and Methods: PCR was carried out using specific primers for the virulence factor of S. typhi, hp and invA, B. anthracis virulence factors, chr and pa, and Y. pestis toxin, pla. The primer sets were tested for cross reactivity with individual DNA strains and mixtures in a multiplex PCR format. For determination of sensitivity of the method, we used colony counting and genomic DNA dilution. Results: Theresult with standard strains revealed that primers are specific for each strain as they have successfully amplified the desire products. The sensitivity analysis showed a 1-10 CFU and 1-10 copies of the genomes. In this respect, the accuracy of the result was checked by sequencing of the 1083-bp PCR products, except for anthrax, which was determined by enzymatic digestion. Conclusion: We have found a rapid, specific, sensitive, and a non-expensive molecular method for detection of three potent human pathogens. This finding provide an efficient diagnostic tool to determine these bacteria in bioterrorism samples. Keywords: Detection, Salmonella typhi, Bacillus antheracis, Yersinia pestis, Multiplex PCR ÖÏźǀţƹŵřŵźųÕÒƽƵŹŚưƃ ÏÎƽƵŹƹŵƱŚŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾƨƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽ ƶƬŬƯ