Multiplexed fuel analysis
Compositions, testing chambers and methods for testing a fuel sample for microbial contamination (including fuels treated with a biocide) are provided, which comprise: a quantity of hydrocarbon fuel; a microbial contamination wherein the microbial contamination further comprises nucleic acid in the form of both DNA, RNA or a combination thereof, and an analyzing solution; wherein the analyzing solution comprises at least six (6) primer pairs for amplification of at least one target locus, wherein at least one primer of each pair of primers is labeled with a fluorescent dye and wherein at least one of the primer pair binds to the nucleic acid of the microbial contamination.
1 . A composition comprising at least seven (7) primer pairs in which at least one primer of each primer pair is labeled with a fluorescent dye;
the first primer of said first primer pair having at least 90% homology to the sequence set forth in SEQ ID NO: 1 and the second primer of said first primer pair having at least 90% homology to the sequence set forth in SEQ ID NO: 2 or 3;
the first primer of said second primer pair having at least 90% homology to the sequence set forth in of SEQ ID NO: 4 and the second primer of said second primer pair having at least 90% homology to the sequence set forth in SEQ ID NO: 5;
the first primer of said third primer pair having at least 90% homology to the sequence set forth in SEQ ID NO: 6; and said second primer of said fifth primer pair having at least 90% homology to the sequence set forth in SEQ ID NO: 7;
the first primer of said fourth primer pair having at least 90% homology to the sequence set forth in SEQ ID NO: 7, and the second primer of said sixth primer pair having at least 90% homology to the sequence set forth in SEQ ID NO: 8;
the first primer of said fifth primer pair having at least 90% homology to the sequence set forth in SEQ ID NO: 9, 10 or 11; and said second primer of said fifth primer pair having at least 90% homology to the sequence set forth in SEQ ID NO: 12;
the first primer of said sixth primer pair having at least 90% homology to the sequence set forth in SEQ ID 39, and the second primer of said sixth primer pair having at least 90% homology to the sequence set forth in SEQ ID 40; and
the first primer of said seventh primer pair having at least 90% homology to the sequence set forth in SEQ ID 45, and the second primer of said seventh primer pair having at least 90% homology to the sequence set forth in SEQ ID 46.
2 . The composition of claim 1 in a solution.
3 . The composition of claim 1 in a lyophilized pellet.
4 . The composition of claim 3 , wherein said lyophilized pellet is in a biochip.
5 . The composition of claim 1 , wherein at least 3 different dyes are used to label the primers.
6 . The composition of claim 1 , wherein at least one primer has:
(i) at least one mismatch to nontarget sequences within 3 nucleotides from the 3′ end creating a primer with one mismatch to nontarget sequences; or
(ii) at least 2 mismatches to nontarget sequences within 3 nucleotides from the 3′ end and wherein said at least 2 mismatches are adjacent with the addition of extra mismatches right next to the original mismatch, creating a primer with one mismatch to target sequences and two consequential mismatches to nontarget sequences; or
(iii) with the at least one nucleotide truncation of primers from the 5′ end to result in primers at least one primer with a Tm of 1-2° C. below the optimized annealing temperature; or
(iv) an at least one nucleotide elongation of the primer at the 5′ end result in a kinetically unfavorable binding complex; or
(v) with an elimination of at least one GC nucleotide pair within the first two positions.
7 . A composition comprising at least seven (7) primer pairs in which at least one primer of each primer pair is labeled with a fluorescent dye;
the first primer of said first primer pair having at least 95% homology to the sequence set forth in SEQ ID NO: 1 and the second primer of said first primer pair having at least 95% homology to the sequence set forth in SEQ ID NO: 2 or 3;
the first primer of said second primer pair having at least 95% homology to the sequence set forth in of SEQ ID NO: 4 and the second primer of said second primer pair having at least 95% homology to the sequence set forth in SEQ ID NO: 5;
the first primer of said third primer pair having at least 95% homology to the sequence set forth in SEQ ID NO: 6; and said second primer of said fifth primer pair having at least 95% homology to the sequence set forth in SEQ ID NO: 7; the first primer of said fourth primer pair having at least 95% homology to the sequence set forth in SEQ ID NO: 7, and the second primer of said sixth primer pair having at least 95% homology to the sequence set forth in SEQ ID NO: 8;
the first primer of said fifth primer pair having at least 95% homology to the sequence set forth in SEQ ID NO: 9, 10 or 11; and said second primer of said fifth primer pair having at least 95% homology to the sequence set forth in SEQ ID NO: 12;
the first primer of said sixth primer pair having at least 95% homology to the sequence set forth in SEQ ID 39, and the second primer of said sixth primer pair having at least 90% homology to the sequence set forth in SEQ ID 40; and
the first primer of said seventh primer pair having at least 95% homology to the sequence set forth in SEQ ID 45, and the second primer of said seventh primer pair having at least 95% homology to the sequence set forth in SEQ ID 46.
8 . The composition of claim 7 , wherein at least one primer has:
(i) at least one mismatch to nontarget sequences within 3 nucleotides from the 3′ end creating a primer with one mismatch to nontarget sequences; or
(ii) at least 2 mismatches to nontarget sequences within 3 nucleotides from the 3′ end and wherein said at least 2 mismatches are adjacent with the addition of extra mismatches right next to the original mismatch, creating a primer with one mismatch to target sequences and two consequential mismatches to nontarget sequences; or
(iii) with the at least one nucleotide truncation of primers from the 5′ end to result in primers at least one primer with a Tm of 1-2° C. below the optimized annealing temperature; or
(iv) an at least one nucleotide elongation of the primer at the 5′ end result in a kinetically unfavorable binding complex; or
(v) with an elimination of at least one GC nucleotide pair within the first two positions of the 3′ end.
9 . A composition comprising at least seven (7) primer pairs in which at least one primer of each primer pair is labeled with a fluorescent dye;
the first primer of said first primer pair having the sequence set forth in SEQ ID NO: 1 and the second primer of said first primer pair having the sequence set forth in SEQ ID NO: 2 or 3;
the first primer of said second primer pair having the sequence set forth in of SEQ ID NO: 4 and the second primer of said second primer pair having the sequence set forth in SEQ ID NO: 5;
the first primer of said third primer pair having the sequence set forth in SEQ ID NO: 6; and said second primer of said fifth primer pair having the sequence set forth in SEQ ID NO: 7; and
the first primer of said fourth primer pair having the sequence set forth in SEQ ID NO: 7, and the second primer of said sixth primer pair having the sequence set forth in SEQ ID NO: 8;
the first primer of said fifth primer pair having the sequence set forth in SEQ ID NO: 9, 10 or 11; and said second primer of said fifth primer pair having the sequence set forth in SEQ ID NO: 12; and
the first primer of said sixth primer pair having the sequence set forth in SEQ ID 39, and the second primer of said sixth primer pair having the sequence set forth in SEQ ID 40; or
the first primer of said seventh primer pair having the sequence set forth in SEQ ID 45, and the second primer of said seventh primer pair having the sequence set forth in SEQ ID 46.
10 . The composition of claim 9 , wherein at least one primer has:
(i) at least one mismatch to nontarget sequences within 3 nucleotides from the 3′ end creating a primer with one mismatch to nontarget sequences; or
(ii) at least 2 mismatches to nontarget sequences within 3 nucleotides from the 3′ end and wherein said at least 2 mismatches are adjacent with the addition of extra mismatches right next to the original mismatch, creating a primer with one mismatch to target sequences and two consequential mismatches to nontarget sequences; or
(iii) with the at least one nucleotide truncation of primers from the 5′ end to result in primers at least one primer with a Tm of 1-2° C. below the optimized annealing temperature; or
(iv) an at least one nucleotide elongation of the primer at the 5′ end result in a kinetically unfavorable binding complex; or
(v) with an elimination of at least one GC nucleotide pair within the first two positions of the 3′ end.
11 . The composition of claim 1 , further comprising at least one species specific primer in which at least one primer of each primer pair is labeled with a fluorescent dye, wherein the species specific primer pair is specific for the species selected from the group consisting of: Hormoconis resinae, Aspergillus penicillioides and Aspergillus fumigatus.