Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-03-28
2004-03-30
Myers, Carla J. (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S023700, C536S024330, C536S023100, C435S006120, C435S091100, C435S091200
Reexamination Certificate
active
06713620
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel oligonucleotide primer for phosphoridyl inositol in
Bacillus cereus
. The present invention also relates to to a method for the detection of
Bacillus cereus
in food.
BACKGROUND OF THE INVENTION
Among the predominantly occurring food borne pathogenic bacteria,
Bacillus cereus
, an opportunistic pathogen has been found to occur abundantly in Indian foods and also cause illnesses like diarrhoea and/or emesis (Rakh et al. 1988). The illness has been attributed to the presence of enterotoxins and other toxins including haemolysins claborated by strains of
B. cereus
. Conventionally,
B. cereus
is detected by its ability to grow on selective plating media containing egg yolk and inability to utilize mannitol. The isolates are further identified by morphological, cultural and biochemical characteristics. (Duguid, 1996).
Advances made in detection methods have led to the use of polymerase chain reaction (PCR) for the specific detection of
B. cereus
. PCR protocols have been developed for the detection of
B. cereus
group of bacteria in pure culture systems and food samples using specific sets of primers.
Reference is made to the work of Schrafts and Griffiths (1995), wherein primers for the cereolysin AB gene (M 24149) of
B. cereus
was designed. The detection limit for
B. cereus
by PCR in artificially contaminated milk samples was 103 CFU/ml without enrichment of the milk.
Reference is made to the works of Agata et. al. (1995) and Mantynen and Lindstrom (1998), wherein primers for the BceT gene was designed and used to study the distribution of the toxin gene in clinical and food isolates of
B. cereus
. Only qualitative observations were made on this work and no quantification has been reported. It was also postulated that the BceT gene could not be targeted to assess the enterotoxic potential of
B. cereus
strains.
Reference is made to the work of Wang et al. (1997), wherein a universal protocol for PCR detection of a number of food borne pathogenic bacteria was devised using haemolysin as the target gene. Detection of toxin producing strains of
B. cereus
was accomplished using these primers, following overnight enrichment of various food samples in a laboratory growth medium. This work provides only qualitative information and quantification was not addressed.
Reference is made to the work of Hsieh et al. (1999), wherein oligonucleotide primers were designed for sphingomyelinase gene and used for the PCR based detection of strains of
B. cereus
group in food samples. These primers can detect 100 cells/gram of the food sample only after an enrichment step for 8 hours indicating poor sensitivity
Reference is made to the work of Yamada et al (1999) disclosing spiked boiled rice sample with varying cell concentrations of
B. cereus
. The rice sample was enriched in nutrient broth for different time intervals. No amplification was observed with non-enriched food samples with a gyrase D specific primers, even when the initial cell number was 10
4
CFU of
B. cereus
per gram of boiled rice. Detection of low numbers of
B. cereus
by PCR was possible only after 15 hours enrichment in nutrient broth.
Reference is made to the work of Tsen et al. (2000), wherein primers were designed for 16s ribosomal RNA (Ribo Nucleic Acid) and used for PCR-based quantification of
B. cereus
spiked in food samples. Target cells ranging from 1 to 9 CFU/g of food sample could be detected only after 8 hours enrichment in brain heart infusion broth supplemented with glucose.
In German Patents DE 19915141 and DE 1991514 the sequences refer to 16s ribosomal RNA (Ribo Nucleic Acid) and gyrase B specific primers used for the detection of
Bacillus cereus.
Reference is made to the work of Schrafts and Griffiths (1995) and Herman et al (1995), wherein a method for the isolation of target DNA from milk samples was devised. This method was elaborate comprising multitude of steps using combination of enzymes, detergents and column chromatography. The method also suffered from lack of sensitivity and could only detect 10
3
CFU/ml.
B. cereus
by PCR using primers for cereolysin AB gene,
Reference is made to the work of Yamada et al. (1999), wherein a protocol for the detection of
B. cereus
from boiled rice was described. The method included pre-enrichment step, two steps of filtration, followed by boiling of the samples prior to use in PCR. It was reported that at zero hour, a moderately high count of 2.4×10
4
CFU of
B. cereus
per gram of boiled rice failed to yield any PCR amplified product. Low numbers of
B. cereus
could only be detected after 15 hours of enrichment
The drawback of all these methods have been non-specific detection of target organism i.e.
B. cereus
, lack of reproducibility, failure to detect all the isolates of
B. cereus
in a food system and lack of sensitiveness to detect low numbers of target organism. Besides, the methods are cumbersome and procedures are lengthy. The problem of formation of spores by
B. cereus
group of organisms makes detection by PCR a difficult proposition. In most of the methods a step of enrichment in a suitable laboratory growth medium is included which may take 8 to 15 hours of incubation for building up of cell numbers which can result in target DNA for use in PCR detection.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide an improved method for the detection of
Bacillus cereus
in foods which obviates the drawbacks detailed above.
Another object of the present invention is to use a primer designed for a conserved region of a specific gene in the target organism.
Still another object of the present invention is to use the designed primer in detecting isolates which belong to
B. cereus
group.
Yet another object of the present invention is to detect
B. cereus
in food systems directly by PCR.
Still another object of the present invention is to use a simple and effective method for the preparation of template DNA (Deoxyribo Nucleic Acid) of the organism directly from the foods.
Yet another object of the present invention is to use PCR conditions specific for the detection of target gene in the organism.
Still another object of the present invention is to detect very low numbers of target organism in the food systems.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a novel oligonucleotide primer for phosphotidyl inositol in
B. cereus
said printer comprising
PI-1 (F) 5′ AGTATGGGGAATGAG 3′
(SEQ ID NO: 1)
PI-1 (F) 5′ ACAATTTTCCCACGA 3′
(SEQ ID NO: 2)
The present invention also refers to method for the detection of
B. cereus
in foods said method comprising using primers specific for phosphotidyl inositol gene in
B. cereus
in a mixed microflora, said primers comprising
PI-1 (F) 5′ AGTATGGGGAATGAG 3′
(SEQ ID NO: 1)
PI-1 (F) 5′ ACAATTTTCCCACGA 3′
(SEQ ID NO: 2)
In one embodiment of the invention, the food matrices for detecting
B. cereus
in milk and cooked rice.
In another embodiment of the invention, template DNA from
B. cereus
in cooked rice is extracted using Triton X-100, 0.5-2%, boiling at 96-100° C. for 3-8 min and treatment with phenol : chloroform in the ratio of 22:21-28.27.
In another embodiment of the invention, the template DNA from
B. cereus
in milk is extracted using diethyl ether:chloroform in the ratio of 1:1-1:3, urea 1.5 3.5 M and sodium dodecyl sulphate in a range of 0.5-2%.
In a further embodiment of the invention, the PCR reaction mixture in a total volume of 25 &mgr;l comprises of Tris HCl: 8-12 mM; KCl 45-55 mM, MgCl
2
: 0.5-3.0 mM; gelatin: 0.005 0.02%; individual deoxynucleoside triphosphates: 150-300 &mgr;M; each specific primer: 30 60 picomoles; Taq DNA polymerase: 0.5-2.0) units and template DNA: 1-3 &mgr;l.
In another embodiment of the invention, detection of
B. cereus
is effected by amplification profile of target gene from an initial denaturation at 90-98° C. for 2-8 min, amplification cycles of 28-40, each cycle w
Aiyagari Ramesh
Arun Chandrashekar
Chakravarathy Varadaraj Mandyam
Nireshwalia Keshava
Padmanabha Padmapriya Banada
Council of Scientific and Industrial Research
Ladas & Parry
Myers Carla J.
Sakelaris Sally
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