MOLECULAR STANDARDS FOR MICROBIAL PATHOGENS
A method for constructing a consensus sequence from a sequence alignment. The consensus sequence may be used to generate molecular standards that may substitute for genomic DNA in various assays. Since a molecular standard cannot have unresolved bases, the method removes less informative sequences to resolve all positions in the alignment. Also includes several sequences from pathogenic waterborne species that were constructed according to the method.
1 . A synthetic nucleic acid molecule having a consensus sequence constructed from an alignment of a plurality of nucleic acid sequences from microbial pathogens, the consensus sequence constructed by a method comprising:
generating a frequency matrix comprising the frequency of each base at each position in the alignment;
creating an information matrix comprising the amount of information provided by each base at each position in the alignment; and
calculating an information score for each of the plurality of nucleic acid sequences;
iterating over each position in the alignment and for each position:
calculating base frequencies and determining a highest frequency base;
if the highest frequency base's frequency is higher than a frequency threshold, assigning the highest frequency base to the consensus sequence; and
if the highest frequency base's frequency is lower than the frequency threshold, removing a base corresponding to a nucleic acid sequence with a lowest information score and returning to (a); and
performing a restriction fragment length polymorphism (RFLP) fingerprint of the constructed consensus sequence in silico.
2 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Cryptosporidium andersoni shown in SEQ ID NO:1.
3 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Cryptosporidium baileyi shown in SEQ ID NO:2.
4 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Cryptosporidium bovis shown in SEQ ID NO:3.
5 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Cryptosporidium canis shown in SEQ ID NO:4.
6 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Cryptosporidium felis shown in SEQ ID NO:5.
7 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Cryptosporidium hominis shown in SEQ ID NO:6.
8 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Cryptosporidium meleagridis shown in SEQ ID NO:7.
9 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Cryptosporidium muris shown in SEQ ID NO:8.
10 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Cryptosporidium parvum shown in SEQ ID NO:9.
11 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Cryptosporidium serpentis shown in SEQ ID NO:10.
12 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Cryptosporidium wrairi shown in SEQ ID NO:11.
13 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Giardia intestinalis shown in SEQ ID NO:12.
14 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Encephalitozoon intestinalis shown in SEQ ID NO:13.
15 . The synthetic nucleic acid molecule according to claim 1 , comprising the 18S rRNA consensus sequence for Enterocytozoon bieneusi shown in SEQ ID NO:14.
16 . A synthetic nucleic acid molecule having a consensus sequence selected from the group consisting of: an 18S rRNA consensus sequence for Cryptosporidium andersoni , as shown in SEQ ID NO:1; an 18S rRNA consensus sequence for Cryptosporidium baileyi , as shown in SEQ ID NO:2; an 18S rRNA consensus sequence for Cryptosporidium bovis , as shown in SEQ ID NO:3; an 18S rRNA consensus sequence for Cryptosporidium canis , as shown in SEQ ID NO:4; an 18S rRNA consensus sequence for Cryptosporidium felis , as shown in SEQ ID NO:5; an 18S rRNA consensus sequence for Cryptosporidium hominis , as shown in SEQ ID NO:6; an 18S rRNA consensus sequence for Cryptosporidium meleagridis , as shown in SEQ ID NO:7; an 18S rRNA consensus sequence for Cryptosporidium muris , as shown in SEQ ID NO:8; an 18S rRNA consensus sequence for Cryptosporidium parvum , as shown in SEQ ID NO:9; an 18S rRNA consensus sequence for Cryptosporidium serpentis , as shown in SEQ ID NO:10; an 18S rRNA consensus sequence for Cryptosporidium wrairi , as shown in SEQ ID NO:11; an 18S rRNA consensus sequence for Giardia intestinalis , as shown in SEQ ID NO:12; an 18S rRNA consensus sequence for Encephalitozoon intestinalis , as shown in SEQ ID NO:13; and an 18S rRNA consensus sequence for Enterocytozoon bieneusi , as shown in SEQ ID NO:14.
17 . A synthetic nucleic acid construct containing the synthetic nucleic acid having a consensus sequence as in claim 16 .
18 . The synthetic nucleic acid construct of claim 17 , wherein the construct is linear.
19 . The synthetic nucleic acid construct of claim 17 , wherein the construct is a circular plasmid.
20 . A microbial pathogen test kit comprising:
a synthetic nucleic acid molecule having a consensus sequence constructed from an alignment of a plurality of nucleic acid sequences from microbial pathogens, the consensus sequence constructed by a method comprising:
generating a frequency matrix comprising the frequency of each base at each position in the alignment;
creating an information matrix comprising the amount of information provided by each base at each position in the alignment; and
calculating an information score for each of the plurality of nucleic acid sequences;
iterating over each position in the alignment and for each position:
calculating base frequencies and determining a highest frequency base;
if the highest frequency base's frequency is higher than a frequency threshold, assigning the highest frequency base to the consensus sequence; and
if the highest frequency base's frequency is lower than the frequency threshold, removing a base corresponding to a nucleic acid sequence with a lowest information score and returning to (a); and
performing a restriction fragment length polymorphism (RFLP) fingerprint of the constructed consensus sequence in silico.