Claims for Patent: 9,238,842

Summary for Patent: 9,238,842

Title:	Method for detecting the genus of Bacillus in samples from oil reservoirs
Abstract:	The invention provides a quick method for detecting the genus of Bacillus in samples from oil reservoirs, which comprise: collecting samples from oil reservoirs; inactivating microbes except for spore-forming Bacillus in the samples through high temperature; collecting spores from the samples; incubating the collected spores in a medium, and stimulating to resurrect Bacillus under in situ temperatures of the oil reservoirs; detecting the resurrected Bacillus in the cultures using molecular biological techniques. The present invention provides an effective method to detect the genus of Bacillus in samples from oil reservoirs, which is conducive to the discovery of the functional Bacillus for microbial enhanced oil recovery (MEOR) and to revealing the ecosystem of Bacillus in oil reservoirs.
Inventor(s):	Zhang; Fan (Beijing, CN), She; Yuehui (Jingzhou, CN), Hou; Dujie (Beijing, CN), Chai; Lujun (Beijing, CN)
Assignee:	China University of Geosciences, Beijing (Beijing, CN)
Application Number:	13/868,561
Patent Claims:	1. A quick method for detecting the genus Bacillus in samples from oil reservoirs, which comprises: (a) collecting a sample from an oil reservoir; (b) inactivating microbes through high temperature; (c) collecting spores from the inactivated sample; (d) incubating the collected spores in an appropriate medium, and stimulating to resurrect the Bacillus under the in situ temperature of the oil reservoir; and (e) detecting the resurrected Bacillus in the cultures using molecular biological techniques, wherein said step of inactivating microbes through high temperature comprises transferring 500 ml of the sample into a sterile flask in a sterile condition, sealing the flask with a sterile ventilate membrane, and placing the flask on an electric heater and boiling the sample for 10 minutes, then cooling the sample at room temperature, wherein said step of collecting spores from the inactivated sample comprises, after the cooling step, transferring the inactivated sample into 50 ml sterile centrifuge tubes in a sterile condition and centrifuging at 10,000.times.g for 15 minutes to produce a supernatant and a pellet, transferring the supernatant to a sterile container for medium preparation, resuspending the pellet with 1 ml sterile water, and combining the suspension from all the centrifuge tubes into a 10 ml mixture, the mixture containing spores, wherein said medium is compatible with the environment from which the sample was collected, and can provide nutrients for stimulating spores, the medium prepared by solving 2 g molasses, 0.16 g (NH.sub.4).sub.2HPO.sub.4, 0.2 g KNO.sub.2 and the supernatant from the collecting step to obtain a 100 ml solution, followed by adding 2 g crude oil from the reservoir from which the sample was collected to the 100 ml solution, and autoclaving at 121.degree. C. for 20 minutes. 2. The quick method for detecting the genus Bacillus in samples from oil reservoirs according to claim 1, wherein the method is carried out with samples from various oil reservoirs with different geological conditions and exploitation methods. 3. The quick method for detecting the genus Bacillus in samples from oil reservoirs according to claim 2, wherein said samples are selected from samples collected directly from the wellheads of injection wells, oil-water samples collected from production wells, formation water samples, oil-water flowing-back samples of the oil wells, and core samples from oil reservoirs. 4. The quick method for detecting the genus Bacillus in samples from oil reservoirs according to claim 3, wherein said step (a) of collecting a sample comprises: collecting an oil-water sample and placing the sample into sterile plastic bottles, full-filled, sealing the plastic bottles and recording the in situ temperature of the oil reservoir from which the sample was collected. 5. The quick method for detecting the genus Bacillus in samples from oil reservoirs according to claim 1, wherein said step (d) of incubating the collected spores in a medium, and stimulating to resurrect the Bacillus under the in situ temperature of the oil reservoir comprises: incubating the 10 ml mixture collected in step (c) in a 100 ml flask with 30 ml sterilized medium, sealing the cover of the flask, cultivating the total 40 ml mixture for 7-15 days at 150 rpm at the in situ temperature of the oil reservoir from which the sample was collected, to resurrect the Bacillus and obtain resurrected culture, wherein said in situ temperature of the oil reservoir is from 10.degree. C. to 110.degree. C. 6. The quick method for detecting the genus Bacillus in samples from oil reservoirs according to claim 5, wherein said step of detecting the resurrected Bacillus in the cultures using molecular biological techniques comprises: transferring 5 ml of the stimulating medium in the step (d) into a sterile centrifuge tube in a sterile condition, centrifuging at 10,000.times.g for 10 minutes for cell collection, removing the supernatant and adding 1 ml distilled water to resuspend the pellet; extracting DNA in the resuspended pellet and amplifying the DNA by PCR using specific 16S RNA gene primers for bacteria to construct a clone library, and sequencing and analyzing the representative clones to determine their phylogenetic affiliations.

Details for Patent 9,238,842

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	ZOSTAVAX	zoster vaccine live	For Injection	125123	05/25/2006		2032-11-30