TABLE 2.

Detection of TG-1 bacteria in various habitats using the new designed primer sets for specific amplification of major lineages in the phylum^a

Habitat or strain	Detection of TG-1 bacteria withb,c:
	Primer set 1	Primer set 2	Primer set 3	Primer set 4
Lahn River sedimentd	−	−	−	−
Marburg forest soild	−	−	−	−
Italian rice soile	−	6/0 (14)	−	−
Cow rumenf	−	−	5/0 (16)	−
Reticulitermes santonensis gutg	+	3/3 (16)	5/3 (17)	−
Zootermopsis nevadensis gutg	+	3/3 (19)	5/2 (12)	−
Pachnoda ephippiata gutg	−	−	±	−
Strain Pei191h	−	−	+	−

^aSee Table 1.

^b+, PCR product of the expected size; −, no PCR product; ±, results with different preparations varied. If clone libraries were analyzed, the number of TG-1 phylotypes/number of “Endomicrobia” phylotypes among the total clones tested (in parentheses) is indicated.

^cEach reaction mixture (25 μl) contained reaction buffer (Invitrogen), MgCl₂ (see Table 1), deoxynucleoside triphosphates (200 μM each), primers (0.3 μM each), dimethyl sulfoxide (1 μl), DNA extract (200 to 300 ng), and Taq DNA polymerase (1.25 U; Invitrogen). The thermal cycling consisted of an initial denaturation step of 5 min at 96°C, followed by 30 cycles consisting of 30 s at 94°C, 30 s at the annealing temperature (Table 1), and 45 s at 72°C.

^dTopsoil under Fagus sylvatica and anoxic sediment of the Lahn River were freshly collected in Marburg, Germany.

^eDried rice soil (Oryza sativa) from wetland rice fields of the Italian Rice Research Institute in Vercelli (Italy) was regenerated for 3 days as described by Frenzel et al. (5).

^fRumen contents of a freshly slaughtered Holstein cow.

^gPrepared as previously described (13, 21).

^hIsolated from the hindgut of Pachnoda ephippiata.