Molecular standards for microbial pathogens

Abstract

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.

What is claimed is: 
     
         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.