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US 2014/0178883 A1Published application
Method for the identification of propane-oxidizing bacteria
AssigneeENI S.p.A.
InventorsFrancesco RODRIGUEZ, Francesca de Ferra, Elisabetta Franchi
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Abstract
The invention relates to a method for the identification of propane-oxidizing bacteria which is based on the identification of at least one fragment of the prmA gene encoding the alpha subunit of the propane monooxygenase enzyme and/or the prmD gene encoding an ancillary protein involved in the oxidation reaction of propane by gene amplification in the presence of pairs of primers selected in correspondence of homologous portions, deduced from the alignment of the prmA and prmD sequences.
Claims10
(canceled)
A method for identification of propane-oxidizing bacteria comprising:
- extracting a polynucleotide from environmental samples and subsequently identifying at least one fragment of a gene prmA comprising the nucleotide sequence selected from the group consisting of SEQ ID NO:
- 154 to SEQ ID NO:
- 174 and SEQ ID NO:
- 175, and/or of a gene prmD comprising the nucleotide sequence selected from the group consisting of SEQ ID NO:
- 176 to SEQ ID NO:
- 195 and SEQ ID NO:
- 196,
- wherein the identification of said gene fragments is carried out by gene amplification in the presence of pairs of primers selected in correspondence of homologous portions deduced from the alignment of the prmA and prmD sequences.
5 more elements
A method for the identification of propane-oxidizing bacteria in a sample comprising hybridization of a labelled probe with DNA of the sample, wherein the probe consists of at least one sequence selected from the group consisting of the following sequences:
- FORWARD PRIMERS:
- prmA_1F:
- (SEQ ID NO:
- 1)
- CTTCCCGATGGARGARGARAARGA
- XA_0301F:
- (SEQ ID NO:
- 2)
- GCCCATGCGAAGATCACCGA
- XA_0358F:
- (SEQ ID NO:
- 3)
- CCGCTTCGGCACCGACTACAC
- XA_0370F:
- (SEQ ID NO:
- 4)
- ACCGACTACACCTTCGAGAAGGC
- XA_0382F:
- (SEQ ID NO:
- 5)
- TTCGAGAAGGCCCCCAAGAAGGA
- XA_0406F:
- (SEQ ID NO:
- 6)
- CCTCTCAAGCAGATCATGCGGTC
- XA_0930F:
- (SEQ ID NO:
- 7)
- ACGGTCTTCCACTCGGTGCAGTC
- XA_0993F:
- (SEQ ID NO:
- 8)
- TGATGGCGCTCGCCGACGAGCG
- XA_1041F:
- (SEQ ID NO:
- 9)
- CTGCGGTACGCGTGGTGGAACAA
- XA_1089F:
- (SEQ ID NO:
- 10)
- GCACCTTCATCGAGTACGGCAC
- XA_1107F:
- (SEQ ID NO:
- 11)
- CGGCACCAAGGACCGCCGCAAGGA
- XA_1152F:
- (SEQ ID NO:
- 12)
- GGCGGCGGTGGATCTACGACGA
- XA_1170F:
- (SEQ ID NO:
- 13)
- TCATCCCGCTCGAGAAGTACGG
- XA_1233F:
- (SEQ ID NO:
- 14)
- GTCGAGGAGGCGTGGAAGCG
- XA_1305F:
- (SEQ ID NO:
- 15)
- GGCTGGCCGGTGAACTACTGGCG
- XA_1390F:
- (SEQ ID NO:
- 16)
- TCCAAGTACGGCAAGTGGTGGGAG
- XA_1485F:
- (SEQ ID NO:
- 17)
- ACCGGTGCTGGACCTGCATGGT
- XA_1625F:
- (SEQ ID NO:
- 18)
- GGCCGCCCGACCCCGAACATGGG
- XA_460F:
- (SEQ ID NO:
- 19)
- GTGTACGGCGCCATGGACGG
- XA_526F:
- (SEQ ID NO:
- 20)
- CTCGAATGGCAGAAGCTGTTCCT
- XA_586F:
- (SEQ ID NO:
- 21)
- GCGATGCCGATGGCCATCGACGC
- XA_745F:
- (SEQ ID NO:
- 22)
- AAGGCGTTCGCGAACAACTACGC
- XA_789F:
- (SEQ ID NO:
- 23)
- TTCGGTGAAGGCTTCATCACCGG
- prmA_2F:
- (SEQ ID NO:
- 24)
- GGTCGCCGAGACNGCNTTYACNAA
- prmA_49F:
- (SEQ ID NO:
- 25)
- GCGAAGATCACCGAGCTGT
- prmA_733(f):
- (SEQ ID NO:
- 26)
- CGCAATCGTCCGCTGCTC
- XA_16F:
- (SEQ ID NO:
- 27)
- GGCGCACATTGAGTAGGCA
- XA_17F:
- (SEQ ID NO:
- 28)
- TGCAGATGATCGACGAGGT
- XA_18F:
- (SEQ ID NO:
- 29)
- TCGCGGCACATCTCCAACGG
- XA_19F:
- (SEQ ID NO:
- 30)
- CGGACTTCGAGTGGTTCGA
- XA_20Rf:
- (SEQ ID NO:
- 31)
- AACAAGCCGATCGCGTTCG
- XA_21Rf:
- (SEQ ID NO:
- 32)
- CCGAACATGGGCCGGCTCA
- XA_22F:
- (SEQ ID NO:
- 33)
- GCCCGACCCCGAACATGGG
- XA_23Rf:
- (SEQ ID NO:
- 34)
- TGGCAGAAGCTGTTCCTGTCGAT
- XA_24F:
- (SEQ ID NO:
- 35)
- AGCTACGCCGAGATGTGGC
- XA_25Rf:
- (SEQ ID NO:
- 36)
- TGGATCTACGACGACTACTAC
- XA_26F:
- (SEQ ID NO:
- 37)
- GTCCGCGACGACGGCAAGACC
- XA_27Rf:
- (SEQ ID NO:
- 38)
- AAGCAGATCATGCGGTCCTAC
- XA_28F:
- (SEQ ID NO:
- 39)
- GTCCGCGACGACGGCAAGAC
- XA_29F:
- (SEQ ID NO:
- 40)
- TCCGCGGCAACATGTTCCG
- XA_30F:
- (SEQ ID NO:
- 41)
- GCGGTGCAGATGATCGACGA
- XA_31Rf:
- (SEQ ID NO:
- 42)
- GAGATGTGGCGGCGGTGGA
- XA_32Rf:
- (SEQ ID NO:
- 43)
- AACTACTGGCGGATCGACGCG
- XA_33Rf:
- (SEQ ID NO:
- 44)
- GACGGCAAGACCCTGGTC
- Xmo_10F:
- (SEQ ID NO:
- 45)
- TGGTGGAACAACCACTGCGTGGT
- Xmo_11F:
- (SEQ ID NO:
- 46)
- CAGTGGCGGACCTACTGCTCGG
- Xmo_1F:
- (SEQ ID NO:
- 47)
- TGGTTCGAGCACAACTAYCCNGGNTGG
- Xmo_3Rf:
- (SEQ ID NO:48)
- AAGCCGATCGCGTTCGAGGA
- Xmo_4F:
- (SEQ ID NO:
- 49)
- GATACCAGTACCCGCACCG
- Xmo_5Rf:
- (SEQ ID NO:
- 50)
- CAGATGAACCTCAAGAAGCT
- Xmo_6F:
- (SEQ ID NO:
- 51)
- TACATGAACAACTACATCGA
- Xmo_9F:
- (SEQ ID NO:
- 52)
- CAGGAGGCGCACATTGAGTAGG
- Xmo_F:
- (SEQ ID NO:
- 53)
- ACGATCCAGATGAACCTCAAGA
- Xmo_Rf:
- (SEQ ID NO:
- 54)
- TACGCCGAGATGTGGCGGC
- REVERSE PRIMERS:
- XA_30Fr:
- (SEQ ID NO:
- 55)
- ACCTCGTCGATCATCTGCA
- XA_0288R:
- (SEQ ID NO:
- 56)
- GACAACTCGGTGATCTTCGC
- XA_0348R:
- (SEQ ID NO:
- 57)
- GCCTTCTCGAAGGTGTAGTCGGT
- XA_0360R:
- (SEQ ID NO:
- 58)
- TCCTTCTTGGGGGCCTTCTCGAA
- XA_0393R:
- (SEQ ID NO:
- 59)
- CGGGAAGTAGGACCGCATGATCTG
- XA_0408R:
- (SEQ ID NO:
- 60)
- TTCTCTTCCTCCATCGGGAAGTA
- XA_0444R:
- (SEQ ID NO:
- 61)
- GGCACCGTCCATGGCGCCGTA
- XA_0567R:
- (SEQ ID NO:
- 62)
- ACCGCGTCGATGGCCATCGGCAT
- XA_0624R:
- (SEQ ID NO:
- 63)
- TGACGAACCTCGTCGATCATCTG
- XA_0745R:
- (SEQ ID NO:
- 64)
- CCGATGGTGCCCGCGTAGTTGTT
- XA_0779R:
- (SEQ ID NO:
- 65)
- GGTGATCGCGTCGCCGGTAATGAA
- XA_0866R:
- (SEQ ID NO:
- 66)
- TTGGCGGCCGCCTCGTCGGGCAT
- XA_0944R:
- (SEQ ID NO:
- 67)
- GAGTAGCCGTTGGAGATGTG
- XA_0983R:
- (SEQ ID NO:
- 68)
- AGTGGACGGTTGCGCTCGTCGGC
- XA_1073R:
- (SEQ ID NO:
- 69)
- TCCTTGGTGCCGTACTCGATGAA
- XA_1091R:
- (SEQ ID NO:
- 70)
- TCCCGGTCCTTGCGGCGGTCCTT
- XA_1214R:
- (SEQ ID NO:
- 71)
- CGCTTCCACGCCTCCTCGAC
- XA_1327R:
- (SEQ ID NO:
- 72)
- TGTGCTCGAACCACTCGAAGTCC
- XA_1469R:
- (SEQ ID NO:
- 73)
- GCGGGAACCATGCAGGTCCAGCA
- XA_1548R:
- (SEQ ID NO:
- 74)
- GTCCAGTAGCAGGTTTCCGAGCA
- XA_1615R:
- (SEQ ID NO:
- 75)
- CCCGTGAGCCGGCCCATGTTCGG
- XA_1714R:
- (SEQ ID NO:
- 76)
- TGACCGACCAGGGTCTTGCCGTC
- XA_18Fr:
- (SEQ ID NO:
- 77)
- CCGTTGGAGATGTGCCGCGA
- XA_19Fr:
- (SEQ ID NO:
- 78)
- TCGAACCACTCGAAGTCCG
- XA_20R:
- (SEQ ID NO:
- 79)
- CGAACGCGATCGGCTTGTT
- XA_21R:
- (SEQ ID NO:
- 80)
- TGAGCCGGCCCATGTTCGG
- XA_22Fr:
- (SEQ ID NO:
- 81)
- CCCATGTTCGGGGTCGGGC
- XA_23R:
- (SEQ ID NO:
- 82)
- ATCGACAGGAACAGCTTCTGCCA
- XA_24Fr:
- (SEQ ID NO:
- 83)
- GCCACATCTCGGCGTAGCT
- XA_25R:
- (SEQ ID NO:
- 84)
- GTAGTAGTCGTCGTAGATCCA
- XA_26Fr:
- (SEQ ID NO:
- 85)
- GGTCTTGCCGTCGTCGCGGAC
- XA_27R:
- (SEQ ID NO:
- 86)
- GTAGGACCGCATGATCTGCTT
- XA_28Fr:
- (SEQ ID NO:
- 87)
- GTCTTGCCGTCGTCGCGGAC
- XA_29Fr:
- (SEQ ID NO:
- 88)
- CGGAACATGTTGCCGCGGA
- XA_30Fr:
- (SEQ ID NO:
- 89)
- TCGTCGATCATCTGCACCGC
- XA_31R:
- (SEQ ID NO:
- 90)
- TCCACCGCCGCCACATCTC
- XA_32R:
- (SEQ ID NO:
- 91)
- CGCGTCGATCCGCCAGTAGTT
- XA_33R:
- (SEQ ID NO:
- 92)
- GACCAGGGTCTTGCCGTC
- Xmo_10R:
- (SEQ ID NO:
- 93)
- ACCACGAGTAGGTCCGCCACTG
- Xmo_11R:
- (SEQ ID NO:
- 94)
- CCGAGCAGTAGGTCCGCCACTG
- Xmo_2R:
- (SEQ ID NO:
- 95)
- TGCGGCTGCGCGATCAGCGTYTTNCCRTC
- Xmo_3R:
- (SEQ ID NO:
- 96)
- TCCTCGAACGCGATCGGCTT
- Xmo_4Fr:
- (SEQ ID NO:
- 97)
- CGGTGCGGGTACTGGTATC
- Xmo_5R:
- (SEQ ID NO:
- 98)
- AGCTTCTTGAGGTTCATCTG
- Xmo_6Fr:
- (SEQ ID NO:
- 99)
- TCGATGTAGTTGTTCATGTA
- Xmo_Fr:
- (SEQ ID NO:
- 100)
- TCTTGAGGTTCATCTGGATCGT
- Xmo_R:
- (SEQ ID NO:
- 101)
- GCCGCCACATCTCGGCGTA,
- which is complementary to the sequences of a gene prmA of propane-oxidizing bacteria,
- or
- the probe consists of at least one sequence selected from the group consisting of the following sequences:
- FORWARD PRIMERS:
- XD_043F:
- (SEQ ID NO:
- 102)
- TCGTCCACCGAGTTCTCCAACA
- XD_071F:
- (SEQ ID NO:
- 103)
- GTGTCACCTTGATGAACACCCC
- XD_181F:
- (SEQ ID NO:
- 104)
- AACCGGCTCGAGTTCGACTACG
- XD_2Rf:
- (SEQ ID NO:
- 105)
- GTTCTCCAACATGTGCGGCG
- XD_3Rf:
- (SEQ ID NO:
- 106)
- CCGTCGATGATCCGCGTC
- XD_4Rf:
- (SEQ ID NO:
- 107)
- TCTTCGAGGAGATCAGCTCCAC
- XD_5Rf:
- (SEQ ID NO:
- 108)
- GACGCCGCCGAGTACATCGG
- Xmo_8F:
- (SEQ ID NO:
- 109)
- ACCGAGTTCTCCAACATGTG
- XD_6Rf
- (SEQ ID NO:
- 110)
- TTCGAGGAGATCAGCTCCACC
- Xmo_7Rf:
- (SEQ ID NO:
- 111)
- CATGCAATTCGGATCGKCCA
- XD_7F:
- (SEQ ID NO:
- 112)
- GGCTCCATCTTCGAGGAGATCA
- REVERSE PRIMERS:
- prmD_1R:
- (SEQ ID NO:
- 113)
- ATGGACCATCCGNCCRTARTGNGT
- XD_061R:
- (SEQ ID NO:
- 114)
- ACGCGGCCGATCGGGGTGTTCAT
- XD_136R:
- (SEQ ID NO:
- 115)
- TGGCCGTCGACGCGGATCATCGA
- XD_172R:
- (SEQ ID NO:
- 116)
- TCGGTGAGCTCGTCGTAGTCGAA
- XD_235R:
- (SEQ ID NO:
- 117)
- TGGGTGGAGCTGATCTCCTCGAA
- XD_2R:
- (SEQ ID NO:
- 118)
- CGCCGCACATGTTGGAGAAC
- XD_3R:
- (SEQ ID NO:
- 119)
- GACGCGGATCATCGACGG
- XD_4R:
- (SEQ ID NO:
- 120)
- GTGGAGCTGATCTCCTCGAAGA
- XD_5R:
- (SEQ ID NO:
- 121)
- CCGATGTACTCGGCGGCGTC
- XD_6R:
- (SEQ ID NO:
- 122)
- GGTGGAGCTGATCTCCTCGAA
- XD_7Fr:
- (SEQ ID NO:
- 123)
- TGATCTCCTCGAAGATGGAGCC
- Xmo_7R:
- (SEQ ID NO:
- 124)
- TGGMCGATCCGAATTGCATG
- Xmo_8Fr:
- (SEQ ID NO:
- 125)
- CACATGTTGGAGAACTCGGT,
- which is complementary to the gene prmD of the propane-oxidizing bacteria.
504 more elements
A method for identification of propane-oxidizing bacteria:
- extracting DNA from a sample;
- putting the extracted DNA in contact with a pair of primers complementary to a prmA or a prmD gene under conditions which allow the amplification of a fragment of the prmA or prmD gene;
- and
- analyzing the gene amplification product by real time PCR or gel-electrophoresis.
1 more element
A method for quantitative determination of propane-oxidizing bacteria, comprising:
- performing gene amplification in the presence of different quantities of genomic DNA of propane-oxidizing bacteria;
- quantitatively determining a gene amplification product;
- constructing a calibration curve;
- and
- quantitatively determining the genomic DNA in a sample to be analyzed by interpolation.
2 more elements
A method for the identification of the presence of propane-oxidizing bacteria in an environmental sample, based on the identification of prmA and/or prmD genes according to the method comprising:
- extracting DNA from an environmental sample and
- subsequently identifying at least one fragment of a prmA gene comprising the nucleotide sequence 1 selected from the group consisting of SEQ ID NO:
- 154 to SEQ ID NO:
- 174 and SEQ ID NO:
- 175, and wherein the nucleotide sequence 1 is from the prmA gene, and/or at least one fragment of a prmD gene comprising the nucleotide sequence 2 selected from the group consisting of SEQ ID NO:
- 176 to SEQ ID NO:
- 195 and SEQ ID NO:
- 196, and wherein the nucleotide sequence 2 is from the prmD gene,
- wherein the identification of said gene fragments is carried out by gene amplification in the presence of pairs of primers selected in homologous portions (i) of aligned prmA gene sequences encoding the alpha subunit of a propane monooxygenase enzyme and/or (ii) of aligned prmD gene sequences encoding an ancillary protein involved in oxidation of propane.
6 more elements
Dependencies are inferred from the claim text.