PCR primers for the rapid and specific detection of...

Details PCR primers for the rapid and specific detection of... PCR primers for the rapid and specific detection of...

1999-12-09

2001-06-26

Myers, Carla J. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023200, C536S023700, C536S024320, C536S024330

Reexamination Certificate

active

06251607

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to PCR primers designed based on a DNA sequence of a gene encoding malic acid dehydrogenase and a specific DNA of
Salmonella typhimurium
, to a probe used in PCR, and to a PCR method for the rapid and specific detection of
Salmonella typhimurium
in food and clinical specimens.
2. Description of related prior art
Among Salmonellae causing food poisoning and Salmonellosis infection, important Salmonellae include
S. typhimurium, S. typhi
, and
S. enteritidis
, which play a significant role in main food pathogenic bacteria around the world.
Traditionally, the method for detecting
S. typhimurium
comprises steps of pre-culturing, culturing on a selective medium, streak culturing and differentiating on a selective agar medium, biochemical identification of suspected colonies, and serological test, which need a time period of at least 5-7 days that might be too late to be of use for understanding of pathogen in a crisis of food poisoning and salmonellosis infection.
Polymerase chain reaction (PCR) can be rapid and reliable for detecting bacteria and virus in various samples. Among all
Salmonella serovars
, PCR primers useful for detecting
S. typhi
and
S. enteritidis
had been reported in literature, while PCR primers for detecting
S. typhimurium
was rarely seen in literature and patents.
As to the technical level in the state-of-art, related literature and patents can be summarized as follows:
a. Patents associated with the detection of Salmonella: (1) U.S. Pat. No. 5,683,883 (1997) related to PCR primers useful for the detection of all
Salmonella serovars
: (2) U.S. Pat. No. 5,824,795 (1998) related to PCR detections of
S. enteritidis
and
S. bongori
; (3) U.S. Pat. No. 5,714,321 (1998) described nucleotide probes useful for detecting all
Salmonella serovars
; (4) U.S. Pat. No. 5,804,378 (1988) disclosed nucleotide probes useful for detecting related Salmonella genus; (5) U.S. Pat. No. 5,681,716 (1997) related nucleotide sequences useful for the detection of
S. typhi
. As stated above, although there were patents relating DNA probe and PCR methods for detecting Salmonella other than
S. typhimurium
, patent associated with PCR detection of
S. typhimurium
has been rarely seen.
b. Study reports: (1) Olsen et al (1995) reported oligonucleotide probe useful for the detection of Salmonella and
S. typhimurium
, which was designed based on the sequence of a cloned 2.3 Kb DNA fragment, however, this probe was used for detecting DNA-DNA hybridization of
S. typhimurium
; (2) Rahn et al (1992) developed a PCR method for the detection of all
Salmonella serovars
, which method was based on the sequence of invA gene of
S. typhimurium
; (3) Cocolin et al (1998) developed PCR method that could detect 33 serotypes of Salmonella; in combination with hydrolytic analysis by restriction enzyme,
S. typhimurium
could be detected also; (4) Miyamoto et al. (1998) had detected Salmonella including
S. typhimurium
by utilizing RAPD ; (5) Cohen et al. (1996) devised a PCR method based on the sequence of the finA gene of
S. typhimurium
for detecting all
Salmonella serovars
in food samples; (6) Stone et al. (1995) had detected
S. typhimurium
with a PCR-hybridization method that was not a direct PCR detection method; (7) Tuchili et al. (1995) detected chickens infected by
S. gallinarum
or
S. typhimurium
with a PCR method involving the InvA gene, unfortunately, both
Salmonella serovars
were detected; (8) Nastasiru and Mammina(1995) studied the epidemic
S. typhimurium
strains by utilizing a PCR-ribotyping process; (9) Way et al. (1993) detected Salmonella, Shigella,
E. coli
, Citrobacter spp with a multiplex PCR method; (10) in addition, immuno-PCR method had been developed for detecting all serovars Salmonella spp. (Fluit et al 1993; Widjojoatmodjo et al. 1992); (11) Kong et al. (1995) and Chary et al. (1993) reported the detection of
S. typhimurium
in water by using genes of enterotoxin and Aromatase (ARO-A) of
S. typhimurium
, however, its specificity was not further confirmed.
Accordingly, there is a need to provide a method for rapid and specific detection of
S. typhimurium
in food and clinical samples.
SUMMARY OF THE INVENTION
As stated above, one object of the invention is to provide two PCR primers designed based on a DNA sequence of a gene encoding malic acid dehydrogenase and a specific DNA of
Salmonella typhimurium
, respectively.
Another object of the invention is to provide a probe specific for the two primers used in PCR method according to the invention.
Still another object of the invention is to provide a PCR method for the rapid and specific detection of
S. typhimurium
in food and clinical specimens using the two primers and the probe mentioned above.


REFERENCES:
Lu, C.-D. and Abdelal, A.T. Gene 123:143-144, 1993.*
Erlich, H.A. et al. Science 252:1643-1651, Jun. 1991.*
Boyd, E.F. et al. Proc. Natl. Acad. Sci. USA 91:1280-1284, Feb. 1994.*
Zwadyk, P. Zinsser Microbiology, 20th ed., Joklik, W.K. et al, eds., Appleton & Lange, Norwalk, 1992, Chapter 35, p. 556-565.

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