Methods and apparatus for preparing, amplifying, and discriminating multiple analytes

A method for simultaneously and non-preferentially amplifying multiple target nucleic acid sequences from multiple microorganisms, if present, within a sample containing nucleic acids from multiple microorganisms, said multiple target nucleic acid sequences including microorganisms of the Salmonella, Shigella, Campylobacter, Yersinia, and Escherichia coli species, said method comprising the steps of:

• obtaining multiple primer oligonucleotides, each said primer oligonucleotide being complementary to a portion of one strand of a target nucleic acid sequence and specific for one of said multiple microorganisms;
• selecting processing conditions for a simultaneous polymerase chain reaction amplification;
• mixing said sample with said multiple primer oligonucleotides in selected concentrations such that substantially similar amplification efficiencies under the selected processing conditions are achieved for each target nucleic acid sequence present in said sample;
• and
• performing a polymerase chain reaction amplification on said mixture;
• wherein the step of obtaining multiple primer oligonucleotides comprises obtaining SEQ ID NO:3 and SEQ ID NO:4 for use in detecting target nucleic acid sequences from microorganisms of the species Shigella.

A method for simultaneously and non-preferentially amplifying multiple target nucleic acid sequences from multiple microorganisms, if present, within a sample containing nucleic acids from multiple microorganisms, said multiple target nucleic acid sequences including microorganisms of the Salmonella, Shigella, Campylobacter, Yersinia, and Escherichia coli species, said method comprising the steps of:

• obtaining multiple primer oligonucleotides, each said primer oligonucleotide being complementary to a portion of one strand of a target nucleic acid sequence and specific for one of said multiple microorganisms;
• selecting processing conditions for a simultaneous polymerase chain reaction amplification;
• mixing said sample with said multiple primer oligonucleotides in selected concentrations such that substantially similar amplification efficiencies under the selected processing conditions are achieved for each target nucleic acid sequence present in said sample;
• and
• performing a polymerase chain reaction amplification on said mixture;
• wherein the step of obtaining multiple primer oligonucleotides comprises obtaining SEQ ID NO:5 and SEQ ID NO:6 for use in detecting target nucleic acid sequences from microorganisms of the species Campylobacter.

A method for simultaneously and non-preferentially amplifying multiple target nucleic acid sequences from multiple microorganisms, if present, within a sample containing nucleic acids from multiple microorganisms, said multiple target nucleic acid sequences including microorganisms of the Salmonella, Shigella, Campnylobacter, Yersinia, and Escherichia coli species, said method comprising the steps of:

• obtaining multiple primer oligonucleotides, each said primer oligonucleotide being complementary to a portion of one strand of a target nucleic acid sequence and specific for one of said multiple microorganisms;
• selecting processing conditions for a simultaneous polymerase chain reaction amplification;
• mixing said sample with said multiple primer oligonucleotides in selected concentrations such that substantially similar amplification efficiencies under the selected processing conditions are achieved for each target nucleic acid sequence present in said sample;
• and
• performing a polymerase chain reaction amplification on said mixture;
• wherein the step of obtaining multiple primer oligonucleotides comprises obtaining SEQ ID NO:7 and SEQ ID NO:8 for use in detecting target nucleic acid sequences from microorganisms of the species Yersinia.

A method for simultaneously and non-preferentially amplifying multiple target nucleic acid sequences from multiple microorganisms, if present, within a sample containing nucleic acids from multiple microorganisms, said multiple target nucleic acid sequences including microorganisms of the Salmonella, Shigella, campylobacter, Yersinia, and Escherichia coli species, said method comprising the steps of:

• obtaining multiple primer oligonucleotides, each said primer oligonucleotide being complementary to a portion of one strand of a target nucleic acid sequence and specific for one of said multiple microorganisms;
• selecting processing conditions for a simultaneous polymerase chain reaction amplification;
• mixing said sample with said multiple primer oligonucleotides in selected concentrations such that substantially similar amplification efficiencies under the selected processing conditions are achieved for each target nucleic acid sequence present in said sample;
• and
• performing a polymerase chain reaction amplification on said mixture;
• wherein the step of obtaining multiple primer oligonucleotides comprises obtaining SEQ ID NO:9 and SEQ ID NO:10 for use in detecting target nucleic acid sequences from microorganisms of the species E. coli.

A method for simultaneously and non-preferentially amplifying multiple target nuclcic acid sequences from multiple microorganisms, if present, within a sample containing nucleic acids from multiple microorganisms, said multiple target nucleic acid sequences including microorganisms of the Salmonella, Shigella, Campylobacter, Yersinia, and Escherichia coli species, said method comprising the steps of:

• obtaining multiple primer oligonucleotides, each said primer oligonucleotide being complementary to a portion of one strand of a target nucleic acid sequence and specific for one of said multiple microorganisms;
• selecting processing conditions for a simultaneous polymerase chain reaction amplification;
• mixing said sample with said multiple primer oligonucleotides in selected concentrations such that substantially similar amplification efficiencies under the selected processing conditions are achieved for each target nucleic acid sequence present in said sample;
• and
• performing a polymerase chain reaction amplification on said mixture;
• wherein the step of obtaining multiple primer oligonucleotides comprises obtaining SEQ ID NO:1 and SEQ ID NO:2 in concentrations of 200 ng each, SEQ ID NO:3 and SEQ ID NO:4 in concentrations of 30 ng each, SEQ ID NO:5 and SEQ ID NO:6 in concentrations of 50 ng each, SEQ ID NO:7 and SEQ ID NO:8 in concentrations of 75 ng each and SEQ ID NO:9 and SEQ ID NO:
• 10 in concentrations of 40 ng each.

A kit useful in the processing of sample material for amplification of one or more selected target nucleic acid sequences from multiple microorganisms, contained therein and for simultaneously and non-preferentially amplifying multiple target nucleic acid sequences from multiple microorganisms, if present, within said processed sample, said kit comprising:

• an extraction buffer solution, said extraction buffer solution comprising at least one detergent composition and at least one salt composition, said salt composition being present in a greater than 1 molar concentration;
• and
• multiple primer oligonucleotides, each said primer oligonucleotide being complementary to a portion of one strand of a target nucleic acid sequence and specific for one of said multiple microorganisms and being present in a selected concentration such that substantially similar amplification efficiencies are achieved for each target nucleic acid sequence present in said sample during amplification under selected processing conditions;
• wherein said extraction buffer solution comprises 1% p-tert-octylphenoxypolyethoxyethanol detergent, 0.5% polyoxyethlenesorbitan monolaurate detergent, and 2.0M NaCl.