Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-27
2001-09-11
Horlick, Kenneth R. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024330
Reexamination Certificate
active
06287771
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to primers capable of hybridization with keratin gene or &agr;
S1
-casein gene. The primers of the present invention can be used for the test of mastitis of mammals such as cows and determination of synthetic level of milk protein in mammary gland cells.
BACKGROUND OF THE INVENTION
Mastitis is an inflammatory disease of mammary tissue caused by infiltrated pathogenic microorganisms in the breast, and causes great economic loss in dairy farming such as decreased milk quantity and selection and screening of infected cows. Fundamental countermeasures to mastitis include early diagnosis and treatment depending on the determination of increased number of somatic cells in milk and PL test in general. These determinations depend on the increased number of somatic cells and tendency to exhibit alkaline pH in milk due to the infection. However, these factors vary greatly between individuals and lactation period, and are deemed not always sufficient for early diagnosis of mastitis. Generally, mastitis milk shows the changes in components with a characteristically decreased content of casein, and a judgment of mastitis using the ratio of nitrogen content in casein and total nitrogen content in milk was tried, however, wide range of the determined values made the judgment unreliable.
On the other hand, the volume of milk or ratio of milk fat have been widely used as indicators to evaluation of milk production of a cow. However, the ratio of milk protein has recently become important in view of the improved production of milk products and/or the trend for better health. Among milk proteins especially casein is considered to be a reliable indicator for evaluation of milk quality. However, conventional method for determination was carried out by separation of casein from milk and measurement of nitrogen content in the separated casein fraction. The procedure is troublesome and requires long period of time without sufficient sensitivity for detailed investigation. Recently, simple determination method of casein content in milk with infrared or near infrared spectroscopy was developed, however, coordination with the observed results with those of conventional methods remain unsolved.
Changes of milk components result from various factors and one presumed mechanism is an infiltration of a causal pathogenic bacteria of mastitis in the mammary gland through lactiferous duct followed by adhesion to epithelial cells of mammary gland resulting the inhibition of biosynthesis of milk components. This means that determination of changes in the ability for production of milk components with mammary gland epithelial cells with some method will provide early detection and diagnosis of mastitis.
The inventors of the present invention aimed to use &agr;
S1
-casein, a most typical protein among milk proteins synthesized in epithelial cells of mammary gland of cow, as an indicator for the level of milk protein synthesis.
Recently to detect the amount of transcription of mRNA a reverse transcription-polymerase chain reaction (RT-PCR) method using a polymerase chain reaction (PCR method: U.S. Pat. Nos. 4,683,195 and 4,683,202) has been developed. The method can be applied for a sensitive qualitative and quantitative determinations of an expressed protein in cells. However, extraction of RNA from biopsied mammary tissue of a cow for the diagnosis of mastitis is practically inapplicable for industrial cow animals and no method has been reported to extract RNA from mammary gland epithelial cells without invasion.
Extraction of RNA from milk demands minimum decomposition of RNA with a ribonuclease. In addition, the number of mammary gland epithelial cells is much smaller than those of other cells and to minimize the influence of milk component on RNA extraction, centrifugation of milk to precipitat cells prior to the extraction is indispensable.
Keratin gene is a cytoskeleton gene which specifically expresses only in epithelial cells including various subtypes and differently express in species and sites. Thus, the primer used for an indicator of the amount of bovine mammary gland epithelial cells requires hybridization with keratin gene which expresses in bovine mammary gland epithelial cells and a correlation between the expressed amount of keratin gene and number of epithelial cells is necessary. Furthermore, &agr;
S1
-casein is known to have four genetic variations and a primer capable of hybridization with any one of a &agr;
S1
-casein variants is required for universal detection method.
DISCLOSURE OF THE INVENTION
The inventors of the present invention have earnestly investigated a method for preparation of RNA from milk and an accurate detection method of mastitis using the resultant RNA under these circumstances, and found a method for preparation of RNA derived from mammary gland epithelial cells in milk by rapid cooling of milk to ice temperature, dilution with a buffer followed by centrifugation to give cellular pellets. The procedure may be repeatedly carried out, if necessary, and RNA is extracted from the resultant cellular pellets. A primer capable of partial amplification of the keratin gene designed to give size of 274 bp in consideration of the amplification range of reactivity in PCR was prepared using a basic (type 2) component 3 keratins. Also, a primer capable of partial amplification of the &agr;
S1
-casein gene designed to give a size of 351 bp in consideration of the amplification range of reactivity in a conserved sequence of the genetic variants of &agr;
S1
-casein gene was synthesized according to conventional methods, and a reverse transcription-polymerase chain reaction [RT-PCR; Wang et al., Proc. Natl. Acad. Sci., USA, 86, 9717-9721 (1989)] using these primers was performed to simply, quickly and accurately detect mastitis and accomplished the present invention.
Therefore, one object of the present invention is to provide a novel DNA for preparation of primers capable of hybridization with keratin gene and &agr;
S1
-casein gene.
The other object of the present invention is to provide a process for amplification of keratin gene and/or casein gene using the primers.
Further object of the present invention is to provide a method for the test and diagnosis of mastitis using the process for amplification of keratin gene and/or casein gene, or a process for determination of synthetic level of milk protein with mammary gland cells. That is, the present invention relates to primers shown by SEQ ID NO: 1-4.
Further, the present invention relates to a primer which hybridizes with keratin gene expressed by SEQ ID NO: 1 or 2. In addition, the present invention relates to a primer which hybridizes with &agr;
S1
-casein gene expressed by SEQ ID NO: 3 or 4.
Furthermore, the present invention relates to a process for amplification of keratin gene and/or casein gene contained in milk by a reverse transcription-polymerase chain reaction (RT-PCR) using these primers.
Said amplification of the present invention is carried out by rapid cooling of milk to temperature of ice, dilution with a buffer, centrifugation, extraction of RNA from the formed precipitates or pellets, followed by amplification of keratin gene and/or casein gene using the RNA as a template and the primers.
Furthermore, the present invention relates to a process for estimation of the ratio of expressed &agr;
S1
-casein gene and keratin gene in milk using these gene amplification methods, and a method for the test of morbidity of mastitis or the level of productivity of milk protein with mammary gland cells.
As described above, the present invention relates to the primers shown in SEQ ID NO: 1-4 in the Sequence Listing. The primer hybridizes with keratin gene and has a sequence of CAG-TGT-CGG-GGG-CTC-TGG (SEQ ID NO: 1 or ATG-TGG-GTC-TGC-GAT-AGG-CT? (SEQ ID NO. ). Also, the primer hybridizes with &agr;
S1
-casein gene and has sequence of GTC-AAG-TGA-ATT-CTG-AGG (SEQ ID NO: 3) or TGG-CAC-TTA-CAG-GAG-AAG SEQ ID NO: 4).
These oligonucleotides are designed in consideration of the d
Imamura Mio
Itagaki Yasuharu
Tanimoto Morimasa
Darby & Darby
Horlick Kenneth R.
Snow Brand Milk Products Co. Ltd.
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