DESC:FORM – 2

THE PATENTS ACT, 1970
(39 OF 1970)

COMPLETE SPECIFICATION
(See Section 10; Rule 13)

“A MICROFLUIDIC KIT FOR DETECTION OF SUBCLINICAL MASTITIS”

SRISTI INNOVATIONS
A Sec 8 company Registered under Companies Act, 1956 having its address at
AES Boys Hostel Campus, Navrangpura,
near Gujarat University Library & SBI Bank,
Ahmedabad, Gujarat 380009.

The following specification particularly describes the nature of the invention:

FIELD OF INVENTION:

The present invention relates to a microfluidic kit for Detection of Subclinical Mastitis. In particular the present invention relates to device for rapid, affordable, quantitative and easy paper based detection of subclinical mastitis.

BACKGROUND AND THE PRIOR ART OF INVENTION:
Mastitis is the inflammatory reaction, of the udder tissue due to physical trauma or microorganisms infections. The microorganism responsible for this is S. aureus. Mastitis is a potentially a fatal mammary gland infection that is common in dairy cattle worldwide. In mastitis the milk from the cow suffering from mastitis has increased somatic cell count and the consequences of mastitis comprises of reduced milk both in terms of quality and quantity, disapproval of milk due to antibiotic remains and increased veterinary expenses. Decrease in productivity of milk, increased mortality, and other expenses for veterinary services, diagnostics and treatment of mastitis result in huge economic losses every year.
The normal pH of milk is 6.4 to 6.7 pH that is increased in the mastitis milk as a result of infiltration of various ions (movement of sodium chloride and sodium bicarbonate). Thus, greater amount of chlorides results in alkaline milk. The alkaline milk due to mastitis is a very good indicator of the disease.
Prevention and control of mastitis requires consistency in sanitizing the cow barn facilities, proper milking procedure and segregation of infected animals. And the Treatment of the disease is carried out by penicillin injection in combination with sulfur drug. However, in most of the cases the severity of the disease cannot be detected since there are no methods available that accurately detect the severity of mastitis.
The most common way to detect mastitis worldwide at the farm level is by California Mastitis Test (CMT). The CMT estimates the amount of DNA present in the milk of the cattle, to detect the amount of WBC in the milk. Said CMT reagent lyses the cells and gels the DNA and the degree of gel formation is used to estimate the number of WBCs in the milk sample. Said CMT is helpful in detecting subclinical mastitis and, although accurate, it serves a little purpose in the detection of the acute clinical mastitis. Since the reaction disappears within 20 seconds, the test must be read quickly and is difficult to interpret. Moreover, this test is generally carried out by veterinary doctors. Thus the said method of detection is accurate and fast however the said conventional method is difficult to use, and difficult to read.
Using as a biomarker for mastitis detection several test kit are reported PortaSCCTM detects the presence of white blood cells in the milk through enzyme esterase assay. Though the test is affordable it lacks sensitivity. Using as a biomarker for mastitis detection several test kit are reported. Pemberton et al. reported the electrochemical detection of enzyme N-acetylglucosaminidase (NAGase) this enzyme is released from injured epithelial cells of mammary tissue into milk. In another study, Welbeck et al., 2011 reported a SPR sensor where NAGase enzyme can be detected. However this kit is required laboratory settings and is not suitable for on farm testing. Tan et al., reported the electrochemical sensor immobilized with antibodies anti-Hp for detection of mastitis milk. This cannot be tested directly from milk but requires a pre-treatment procedure i.e. removal of fat. Peedel and Rinken proposed an immune biosensing system, based on the0020specific binding of S. aureus and the immobilized fluorescence marker helped in detection of S. aureus bacteria in milk within 17 minutes. Garcia-Cordero et al., developed a portable microfluidic sedimentation cytometer for the analysis of Somatic cells. A lab-on-a-chip (LoC) device was developed by Choi et al., for simultaneous detection of microorganisms, antibiotic residues, somatic cells, and pH in raw milk. The chip required the use of microscope for bacterial detection and could detect only high bacterial concentrations.

Therefore there is a need of a detection kit that can accurately detect the severity of the mastitis and does not require to be handled by a veterinary doctor but can be used by the farm owner.

DISADVANTAGES OF THE PRIOR ART:
The invention mentioned in the prior art suffers from all/ any of the below mentioned disadvantages:
1. Most or all of the prior arts fails to provide a microfluidic detection kit that are rapid as well as accurate.
2. Most of them fail to provide on-farm paper based microfluidic kit for detection of subclinical mastitis.
3. Most or all of the prior arts provides a microfluidic detection kit that are pH-based test strips that indicates only yes or no state of the disease with less sensitivity. They do not provide quantitative results.
4. Most or all of the prior arts fails to provide a microfluidic detection kit for detection of subclinical mastitis.
5. Most or all of the prior arts fails to provide a microfluidic detection kit that is rapid and provide results of detection in less than 1 minute.
6. Most or all of the prior arts provides a microfluidic detection kit that involve laboratory setup for detection of subclinical mastitis which is makes them time consuming, technically complex and thereby expensive and non-user friendly.
7. Most or all of the microfluidic kits are not appropriate for on farm testing.
8. Most or all of the prior arts fails to provide a microfluidic detection kit that has long shelf life thereby making it non affordable.
9. Most or all of the prior arts provides a microfluidic detection kit that requires significant amount of milk for testing.
10. Most or all of the prior arts provides a microfluidic detection kit that requires technical expertise and cold chain.

Thus, there is an unmet need to obviate the disadvantages associated with the prior art and come up with an effective solution.

OBJECTS OF INVENTION:
• The main object of the invention is to provide Microfluidic kit for the Detection of Subclinical Mastitis which is rapid, accurate and easy on-farm paper based microfluidic device for detection of subclinical mastitis.
• Another object of the present invention is to provide Microfluidic kit for Detection of Subclinical Mastitis which is quantitative paper based microfluidic kit for detection of subclinical mastitis.
• Yet another object of the present invention is to provide a portable Microfluidic kit for Detection of Subclinical Mastitis.
• Yet another object of the present invention is to provide a Microfluidic kit for Detection of Subclinical Mastitis that provides detection results in less than 1 minute thereby making the detection process fast.
• Yet another object of the present invention is to provide a Microfluidic kit for Detection of Subclinical Mastitis that has long shelf life.
• Yet another object of the present invention is to provide a Microfluidic kit for Detection of Subclinical Mastitis that provides the result in the form of quantitative values thus making the detection process accurate and user-friendly.
• Yet another object of the present invention is to provide a Microfluidic kit that detects mastitis through pH of the milk.
• Yet another object of the present invention is to provide a Microfluidic kit for Detection of Subclinical Mastitis that provides stable result even at high temperatures like 50°C, thus providing a kit that is accurate even at high temperatures.
• Yet another object of the present invention is to provide a Microfluidic kit for Detection of Subclinical Mastitis that is paper based and do not require involvement of laboratory thereby it becomes very user friendly, rapid and affordable and appropriate for on farm testing

Brief Description of Drawing:
Fig. 1 Shows the present microfluidic kit for the detection of Subclinical Mastitis.

Meaning of reference numerals of the present detection kit (P)
Mp : Microfluidic paper
S : Sample zone
D : Detection zone
R : Reference zone

SUMMARY OF THE PRESENT INVENTION:
The present invention provides a microfluidic kit for the detection of mastitis. The present detection kit detects mastitis in less than a minute hence the present invention is rapid and accurate. An aspect of the present invention is to provide a detection kit that detects the severity of the infection on the basis of the pH o milk from cattle. Since the pH of milk is mild acidic that shifts to basic when the cattle is infected with mastitis. Hence even a minute change in the pH of milk helps in the detection as well as the severity of infection. Therefore the present invention provides a detection kit that is sensitive to change in pH of the milk.
The embodiment of present invention is to provide a detection kit that is utilized to detect the severity of mastitis in cattles. The milk sample from the cattle is added to kit after the addition of the indicator, the milk sample reacts with the indicator solution and develops color based on the basicity of the milk. The developed color of the sample solution is compared with the reference color to detect the severity of the infection. Thus the present invention provides with a detection kit that is easy to use, cost effective and accurate.
The present detection kit (P) comprises of:
• Microfluidic paper (Mp)
• Reference zone (R)

DETAILED DESCRIPTION OF THE PRESENT INVENTION:
The embodiment of the present invention is to provide a solution to one or more above mentioned technical problems that are recognized by the inventor of the present invention to accurately detect the mastitis in cattle. The drawings accompanying the description of the complete specification illustrate one or more embodiments of the present invention.
The present detection kit (P) does not require heavy machinery and skilled individuals to carry out the detection of mastitis, thus the present detection kit (P) is user friendly and cost effective. The milk of the infected cattle is basic in nature, hence the present detection kit (P) utilizes pH sensitive indicators for the accurate and rapid detection of the pH of the milk. Therefore the present invention provides a pH sensitive detection kit.
The normal pH of the milk ranges from 6-6.7pH, that turns alkaline when the cattle is suffering from mastitis due to the infiltration of sodium chloride and sodium bicarbonate. The greater amount of chloride results in alkaline milk that is used to detect the infection and the severity of the infection. The present detection kit (P) provides stable results even at 50°C, hence provides a detection kit that is efficient and accurate at high temperatures.
Referring to fig. 1 the present detection kit (P) comprises of:
o A Microfluidic paper (Mp)
o A Reference zone (R)

Said microfluidic paper (Mp) of the present detection kit (P) is a Whatman qualitative filter paper. Said microfluidic paper (Mp) is the surface where the process of detection of the infection takes place.
Said microfluidic paper (Mp) comprises of:
• A Sample zone (S)
• Detection zone (D)
Said sample zone (S) is the zone, where the milk from the infected cattle is added. In said sample zone (S) 30-40µl of milk sample is preferably added. Since the microfluidic paper (Mp) is made up of whatman paper, the sample milk travel towards the detection zone (D) that is present on the both side of the sample zone (S). Said detection zone (D) is the zone, where the sample develops color by reacting with the indicator solution. In the present detection kit (P), the indicator solution is added to the detection zone (D) prior to the addition of the sample in the sample zone (S).
Said indicator solution detects the severity of the infection by reacting with the milk sample and developing color according to the basicity of the milk sample. Said indicator solution consists of pH indicators, biopolymers and color developers. The pH indicator is a combination of methyl red and bromothymol blue that preferably used in ratio of 1:5. Said methyl red is preferably being used in 0.01% molecular weight and bromothymol blue being used in 0.5% molecular weight. Milk from the infected cattle is basic in nature as a result of infiltration of various ions (movement of sodium chloride and sodium bicarbonate).
Said biopolymer being used here is preferably chitosan with 0.5% molecular weight. Said chitosan act as a protectant that substantially increases the stability of color developed and provides uniform distribution of the color developed due to increase in the wet strength of the microfluidic paper (Mp) thus substantially decreases the chances of misinterpretation of the result. In addition the chitosan has antibacterial properties that causes bacterial cell lysis and triggers the release of the intracellular components and finally to cell death. Thus ensuring the causing the color change respective to the degree of infection.
Said color developer used in the the indicator solution is tetrazolium salt. Said tetrazolium salt preferably being used here is Iodonitrotetrazolium chloride (INT) in the range of 5-10mg/ml. Said color developers attaches to the bacteria present in the mastitis milk and changes color from almost colorless to red-purple upon capturing electrons from microorganisms. This allows direct eye visualization of the color change of the detection zone containing bacteria. Said indicator solution comprises of chitosan, Iodonitrotetrazolium chloride, methyl red and bromothymol blue in the ratio of 0.5:0.5:1:5. Said chitosan, color developer and pH indicator being used in the said ratio offer a synergistic action, supporting the development of color with respect to different level of severity of the infection and also increasing the physical properties of the microfluidic paper (Mp).
Once the milk sample reaches the detection zone (D) it reacts with the indicator solution and develops color according to the severity of the infection. The developed color is compared with the reference color provided in the reference zone (R). Said reference zone (R) has reference color scale ranging from 0, 1+, 2+, 3+, 4a+ and 4b+. The color intensity and the scale in the reference zone (R) directly proportional to the CMT measure. And an increase in the CMT measure is directly proportional to the inflammation, thus the increase in the reference color scale indicates the increase in the severity of the infection.

WORKING OF THE PRESENT INVENTION:
The present invention provides a detection kit (P) to detect the mastitis infection and the severity of the infection. Said detection kit (P) provides rapid and accurate results for the detection of infection. The present detection kit measures the increase in the pH of the milk to detect the severity of the infection. In addition unlike the prior art the present detection kit (D) showed good stability. The present detection kit (P) showed good stability even when kept at accelerated temperature 50°C (0 – 150 minutes) before testing with infected milk samples. Thus the detection kit is efficient and provides accurate results even at high temperatures.
Firstly indicator solution is added to the detection zone (D) that comprises of chitosan, Iodonitrotetrazolium chloride, methyl red and bromothymol blue in the ratio of 0.5:0.5:1:5. Said indicator solution plays a vital role in reacting with the milk sample and developing color according to the severity of the infection. Thereafter the milk sample from the infected cattle is taken and is added to the sample zone (S) of the present detection kit (P) in the preferable range of 30-40µl. Since the said microfluidic paper (P) is made of whatman paper the added milk sample starts travelling towards the detection zone of the present detection kit (P). The milk sample reacts with the indicator solution present in the detection zone (D). Said indicator solution sows the synergistic effect in causing the color change with respect to the degree of infection.
Once the color is developed, it is compared with the reference color scale present in the reference zone (R). The reference color scale ranges from 0, 1+…. 4b+. The color on the detection zone (D) intensifies with the increase in the basicity of the milk sample. The reference color scale in the reference zone (R) is directly proportional to the CMT measure. The CMT measure increases with the increase in the inflammation, thus the increase in the reference color scale indicates the increase in the severity of the infection. Therefore reference color scale 4b+ indicates that the cattle is severely infected with mastitis infection.

ADVANTAGES OF INVENTION:
• The present invention provide on-farm paper based microfluidic kit for detection of subclinical mastitis.
• Along with the detection of the infection, the present invention detects the severity of the infection.
• The present invention provides a detection kit that is rapid and the severity of the infection is detected within a minute.
• The present detection kit is user-friendly and can also be used by farmers.
• The present invention provides a paper-based detection kit, thus the present detection kit is environment friendly and cost effective.
• The present detection kit can withstand high temperatures and thus has long shelf life.
• The present detection kit is suitable for on farm testing, thus the samples are not required to be tested in labs, thus the present detection kit time saving.
• The present detection kit does not require technical expertise and cold chain and can be used by anyone.
• The present invention provides a stable results at 50°C, thus the present detection kit is efficient.
,CLAIMS:We claim:
1. A microfluidic kit for detection of subclinical mastitis, wherein the kit (P) comprises of:
• A Microfluidic paper (Mp),
• A Reference zone (R);
Wherein,
Said microfluidic paper (Mp) of the present detection kit (P) is a Whatman qualitative filter paper; said microfluidic paper (Mp) provides a surface for the detection of infection; said microfluidic paper further comprises of:
• A sample zone (S) and,
• A detection zone (D);
Said sample zone (S) is in connection with the detection zone (D) on either side and facilitates the addition of infected milk sample; said detection zone (D) detects the severity of the mastitis infection with the help of an indicator solution that is added on said detection zone (D) prior to the milk sample addition; said indicator solution reacts with the milk sample and develops a color based on the severity of the infection; said indicator solution is a mixture of chitosan, pH indicator, and color developer to facilitate detection process; the developed color is compared with the reference color scale present in the reference zone (R); said reference zone (R) comprises of reference color scale ranging from 0, 1+, 2+, 3+, 4a+ and 4b+; the intensity of the developed color and the color reference scale are directly proportional CMT measure and inflammation due to the infection.
2. A microfluidic kit for detection of subclinical mastitis (P) as claimed in claim 1; wherein said pH indicator is a combination of methyl red (0.01% molecular weight) and bromothymol blue (0.5% molecular weight) that preferably are used in ratio of 1:5
3. A microfluidic kit for detection of subclinical mastitis (P) as claimed in claim 1; wherein said chitosan is being used in the in 0.5% molecular weight.
4. A microfluidic kit for detection of subclinical mastitis (P) as claimed in claim 1; wherein said color developer used is tetrazolium salt preferably being used here is Iodonitrotetrazolium chloride (INT) in the range of 5-10mg/ml.
5. A microfluidic kit for detection of subclinical mastitis (P) as claimed in claim 1; wherein said chitosan, color developer and pH indicator and in the indicator solution are preferably being used in the ratio of 0.5:0.5:1:5.

Dated this 08th Day of April, 2022.

Ms Gopi Trivedi
Authorized Agent of Applicant
IN/PA 993 15

To,
The Controller of Patents
The patent office
At Mumbai