Systems for the detection of targeted gene variations and viral genomes and methods of producing and using same

A method of detecting a target nucleotide sequence in a subject, the method comprising:

• preparing a reaction mixture comprising:
• a biological sample from the subject;
• a forward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 3, 8, 13, 18, 23, 28, 33, 37, 42, 47, 52, 57, 62, and 67;
• a backward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 4, 9, 14, 19, 24, 29, 34, 38, 43, 48, 53, 58, 63, and 68;
• a DNA polymerase with strand displacing activity;
• dNTPs;
• a pH indicator;
• and
• a reaction buffer;
• incubating the reaction mixture to produce an incubated mixture;
• and
• measuring one or more of turbidity, fluorescence, and colorimetric change of the incubated mixture to determine a result, wherein:
• a change in the one or more of turbidity, fluorescence, and colorimetric change indicates a positive result, and
• a positive result indicates the presence of the target nucleotide sequence.

A nucleic acid screening kit to detect the presence of a target nucleotide sequence in a subject, the nucleic acid screening kit comprising:

• a first reaction tube comprising:
• a forward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 3, 8, 13, 18, 23, 28, 33, 37, 42, 47, 52, 57, 62, and 67;
• a backward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 4, 9, 14, 19, 24, 29, 34, 38, 43, 48, 53, 58, 63, and 68;
• a DNA polymerase with strand displacing activity;
• dNTPs;
• and
• a pH indicator;
• a positive control tube comprising:
• a forward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 3, 8, 13, 18, 23, 28, 33, 37, 42, 47, 52, 57, 62, and 67;
• a backward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 4, 9, 14, 19, 24, 29, 34, 38, 43, 48, 53, 58, 63, and 68;
• a DNA polymerase with strand displacing activity;
• dNTPs;
• a pH indicator;
• and
• a synthetic nucleotide sequence substantially identical to the target nucleotide sequence;
• and
• a negative control tube comprising:
• a forward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 3, 8, 13, 18, 23, 28, 33, 37, 42, 47, 52, 57, 62, and 67;
• a backward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 4, 9, 14, 19, 24, 29, 34, 38, 43, 48, 53, 58, 63, and 68;
• a DNA polymerase with strand displacing activity;
• dNTPs;
• and
• a pH indicator.

A nucleic acid screening kit to detect the presence of a target nucleotide sequence in a subject, the nucleic acid screening kit comprising:

• a first reaction tube comprising:
• a forward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 3, 8, 13, 18, 23, 28, 33, 37, 42, 47, 52, 57, 62, and 67;
• a backward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 4, 9, 14, 19, 24, 29, 34, 38, 43, 48, 53, 58, 63, and 68;
• a forward outer primer comprising a nucleic acid sequence selected from SEQ ID NOS. 1, 6, 11, 16, 21, 26, 31, 35, 40, 45, 50, 55, 60, and 65;
• and
• a backward outer primer comprising a nucleic acid sequence selected from SEQ ID NOS. 2, 7, 12, 17, 22, 27, 32, 36, 41, 46, 51, 56, 61, and 66;
• a DNA polymerase with strand displacing activity;
• dNTPs;
• and
• a pH indicator;
• a positive control tube comprising:
• a forward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 3, 8, 13, 18, 23, 28, 33, 37, 42, 47, 52, 57, 62, and 67;
• a backward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 4, 9, 14, 19, 24, 29, 34, 38, 43, 48, 53, 58, 63, and 68;
• a forward outer primer comprising a nucleic acid sequence selected from SEQ ID NOS. 1, 6, 11, 16, 21, 26, 31, 35, 40, 45, 50, 55, 60, and 65;
• and
• a backward outer primer comprising a nucleic acid sequence selected from SEQ ID NOS. 2, 7, 12, 17, 22, 27, 32, 36, 41, 46, 51, 56, 61, and 66;
• a DNA polymerase with strand displacing activity;
• dNTPs;
• a pH indicator;
• and
• a synthetic nucleotide sequence substantially identical to the target nucleotide sequence;
• and
• a negative control tube comprising:
• a forward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 3, 8, 13, 18, 23, 28, 33, 37, 42, 47, 52, 57, 62, and 67;
• a backward inner primer comprising a nucleic acid sequence selected from SEQ ID NOS. 4, 9, 14, 19, 24, 29, 34, 38, 43, 48, 53, 58, 63, and 68;
• a forward outer primer comprising a nucleic acid sequence selected from SEQ ID NOS. 1, 6, 11, 16, 21, 26, 31, 35, 40, 45, 50, 55, 60, and 65;
• and
• a backward outer primer comprising a nucleic acid sequence selected from SEQ ID NOS. 2, 7, 12, 17, 22, 27, 32, 36, 41, 46, 51, 56, 61, and 66;
• a DNA polymerase with strand displacing activity;
• dNTPs;
• and
• a pH indicator.