QUANTITATIVE REAL-TIME PCR USING ARTIFICIAL SEQUENCES AS A STANDARD SAMPLE AND METHOD FOR THE QUANTIFICATION OF MICROORGANISMS USING THE SAME

摘要

PURPOSE: A quantitative real-time PCR using standard samples as artificial base sequences and a quantitative analysis method of microorganism using the same are provided to absolutely estimate the microorganisms by using the quantitative real-time PCR. CONSTITUTION: A quantitative real-time PCR uses standard samples as artificial base sequences. The artificial base sequences include one or more primers and/or probe parts at a gene which represents microbes which will be detected or quantified. The artificial base sequence includes a pair of primer parts at both ends, and one or more probe parts inside the primer parts. The artificial base sequence randomly includes one or more pairs of primer parts and probe parts inside the sequence and at both ends. The artificial base sequence includes a pair of primers of the sequence number 5(SEQ ID NO:5) and 6 or the probe parts of the sequence number described in 7-16. [Reference numerals] (AA) Preparing standard DNA sequences(e.g. consensus sequence, representative sequence); (BB) Arranging design of primer for checking target genes; (CC) Primer sequence design for primer combination; (DD) Arranging design of multiple probe parts for checking target genes; (EE) Probe sequence design for probe combination; (FF) Checking the existance of secondary structure of primer/probe parts; (GG) Non-formation of the secondary structure; (HH) Formation of secondary structure; (II) Checking GC content and melting temperature; (JJ) Re-design of sequence; (KK) High or low; (LL) Good; (MM) Arbitrary sequence synthesis