**1.2 Advanced PCR technologies**

### **1.2.1 Multiplex PCR**

Multiplex PCR can amplify two or more amplicons in a single PCR reaction. For multiplex PCR, each primer set is designed to amplify its target gene and make a PCR product of certain size to the target gene. To perform a multiplex PCR, the concentration of primers, Mg2+, free dNTPs and polymerase must be optimized to allow synthesis of the genes of interest, And also the PCR reaction temperature parameters must be optimized to the best average for amplicon production for all primer sets. This technique saves time and labor more than one target DNA sequence can be detected in each reaction, It might not be optimal if the PCR products are limited in certain sizes and agarose gel staining with ethidium bromide (John Maurer, 2006). Therefore, it is possible to detect multiple pathogens in a sample with a single PCR test (Panicker *et al*., 2004)

#### **1.2.2 Real-Time PCR**

Real-Time PCR technology is based on the ability of detection and quantification of PCR products, or amplicons, as the reaction cycles progress. Higuchi and colleagues introduced this technology (Higuchi *et al*., 1993) and it became possible by including of a fluorescent dye that binds to the amplicon as it is made (Fig. 2. A).

Initially, a fluorescent dye, SYBR green I (A), was used to detect the amplicons. SYBR green I binds the double stranded, DNA amplicon and fluorescences upon illumination with UV light. In TaqMan PCR (B), the oligoprobe contains a fluorescent marker and chemical group that quenches fluorescent of oligoprobe until the dye is liberated by 3' exonuclease activity of the Taq DNA polymerase (Source http://cafe.naver.com/solgent.cafe?iframe\_url= /ArticleRead.nhn%3Farticleid=38&)

In TanMan PCR, an intact, "internal" fluorogenic oligoprobe binds to target DNA sequence, internal to the PCR primer binding sites. This oligoprobe possesses a reporter dye that will fluorescence and a suppressor dye known as quencher that prevent fluorescent activity via Fluorescence Resonance Energy Transfer (FRET). After each PCR cycle, when the doublestranded DNA products are made, a measure of fluorescence is taken after the fluorogenic probe is hydrolytically cleaved from the DNA structure by exonuclease activity of the *Thermus aquaticus* DNA polymerase (Heid *et al*., 1996; Holland *et al*., 1991). Once cleaved, the probe's fluorescent activity is no longer suppressed (Fig. 2. B). FAM (6-Carboxyfluorescein)

Present commercial detection system for *Salmonella* spp. can be classified into four categories. The first, traditional method which uses culture medium and observe colony morphology formed on it. This requires at least four days and experienced skill to perform biological tests,

The second, Enzyme-Linked Immuno-Sorbent Assay (ELISA) detects certain bacteria using immune reaction between antibody and antigen specific for them. This method is easy to use because it makes color change or forms lines but it can be applied only for those which has specific toxin protein and requires more than 106 CFU / ml for detection which needs 16 hours of incubation. The third, Adenosine triphosphate (ATP) detection kit detects level of bacterial contamination by the amount of ATP in sample. This method can not be used for identification of bacteria because it can only tell including the total amount of ATP from food. This is usually used for comparing hygiene level before and after washing. The fourth, genetic method which is based on PCR is highly specific and sensitive enough to detect 100 CFU / ml of bacteria, but at the same time it can detect even the dead cells after processing

Multiplex PCR can amplify two or more amplicons in a single PCR reaction. For multiplex PCR, each primer set is designed to amplify its target gene and make a PCR product of certain size to the target gene. To perform a multiplex PCR, the concentration of primers, Mg2+, free dNTPs and polymerase must be optimized to allow synthesis of the genes of interest, And also the PCR reaction temperature parameters must be optimized to the best average for amplicon production for all primer sets. This technique saves time and labor more than one target DNA sequence can be detected in each reaction, It might not be optimal if the PCR products are limited in certain sizes and agarose gel staining with ethidium bromide (John Maurer, 2006). Therefore, it is possible to detect multiple pathogens

Real-Time PCR technology is based on the ability of detection and quantification of PCR products, or amplicons, as the reaction cycles progress. Higuchi and colleagues introduced this technology (Higuchi *et al*., 1993) and it became possible by including of a fluorescent

Initially, a fluorescent dye, SYBR green I (A), was used to detect the amplicons. SYBR green I binds the double stranded, DNA amplicon and fluorescences upon illumination with UV light. In TaqMan PCR (B), the oligoprobe contains a fluorescent marker and chemical group that quenches fluorescent of oligoprobe until the dye is liberated by 3' exonuclease activity of the Taq DNA polymerase (Source http://cafe.naver.com/solgent.cafe?iframe\_url=

In TanMan PCR, an intact, "internal" fluorogenic oligoprobe binds to target DNA sequence, internal to the PCR primer binding sites. This oligoprobe possesses a reporter dye that will fluorescence and a suppressor dye known as quencher that prevent fluorescent activity via Fluorescence Resonance Energy Transfer (FRET). After each PCR cycle, when the doublestranded DNA products are made, a measure of fluorescence is taken after the fluorogenic probe is hydrolytically cleaved from the DNA structure by exonuclease activity of the *Thermus aquaticus* DNA polymerase (Heid *et al*., 1996; Holland *et al*., 1991). Once cleaved, the probe's fluorescent activity is no longer suppressed (Fig. 2. B). FAM (6-Carboxyfluorescein)

but it is the only common method authorized throughout the world for now.

or cooking food because of the high sensitivity.

in a sample with a single PCR test (Panicker *et al*., 2004)

dye that binds to the amplicon as it is made (Fig. 2. A).

/ArticleRead.nhn%3Farticleid=38&)

**1.2 Advanced PCR technologies** 

**1.2.1 Multiplex PCR** 

**1.2.2 Real-Time PCR** 

and TAMRA(6-Carboxy-Tetramethyl-Rhodamin) are most frequently used as reporter and quencher, respectively. This PCR is often referred to as 5' exonuclase-based, real-time PCR or TaqMan PCR (Mullah *et al*., 1998).

Fig. 2. Real-Time PCR detection of amplicons
