Diagnostic compositions, elements, methods and test kits for amplification and detection of two or more DNA's using primers having matched melting temperatures

A diagnostic test kit for the amplification of first and second target DNA's comprising, in separate packaging:

• a) an aqueous composition buffered to a pH of from about 7 to about 9, and comprising:
• first and second primers which are specific to and hybridizable with, respectively, first and second nucleic acid sequences which are in opposing strands of a first target DNA and which are separated from each other along said opposing strands by from 90 to 400 nucleotides, and
• third and fourth primers which are specific to and hybridizable with, respectively, third and fourth nucleic acid sequences which are in opposing strands of a second target DNA which is the same as or different from said first target DNA, said third and fourth nucleic acid sequences being different from said first and second nucleic acid sequences and being separated from each other along said opposing strands of said second target DNA by from 90 to 400 nucleotides,
• each of said first, second, third and fourth primers having a T.sub.m within the range of from about 65.degree. to about 74.degree. C., all of said primer T.sub.m 's being within about 5.degree. C. of each other, said first and second primers having nucleotide lengths which differ from each other by no more than 5 nucleotides, and said third and fourth primers having nucleotide lengths which differ from each other by no more than 5 nucleotides, and
• b) at least one additional PCR reagent.