A KIT FOR VISUAL PREGNANCY DIAGNOSIS IN LIVESTOCK AND METHOD OF DETECTION THEREOF

Abstract

The present invention relates to a portable kit for pregnancy detection in livestock using urine sample and its method of use. More specifically, the present invention provides a quick, simple and onsite testing kit for pregnancy estimation in livestock comprising a combination of two solutions acidifying agent and colour developing agent in a vehicle, wherein kit helps to develop a red colour in presence of Abscisic acid (about 10 μg/ml and above) as indication of pregnancy and Yellowish orange colour at lower concentration of Abscisic acid as indication of non-pregnancy. Therefore, a kit provides colour based pregnancy detection in livestock using animal urine which provides ease of testing and immediate results. The portable kit would be particularly useful in field or rural settings where laboratory equipment, chemicals, and refrigerated storage are unavailable.

Claims

1. A kit for colour development for detection of Abscisic acid in urine sample as indication of livestock pregnancy, comprising of: a transparent tube for collection of urine sample from subject animal, a first solution of acidifying agent, a second solution of colour developing agent in a vehicle, droppers with graduations for addition of first and second solutions, a standard colour bands Characterised in that the kit provides a colour development in urine sample solution in presence of first solution and second solution, wherein the colour is red colour in presence of Abscisic acid (about 10 μg/ml and above) as indication of pregnancy and Yellowish orange colour at lower concentration of Abscisic acid as indication of non-pregnancy.

2. The kit as claimed in claim 1, wherein the tube is selected from glass tube and plastic tube.

3. The kit as claimed in claim 1, wherein colour developing agent is selected from parahydroxy benzaldehyde.

4. The kit as claimed in claim 1, wherein the vehicle is sulfuric acid.

5. The kit as claimed in claim 4, wherein normality of sulfuric acid vehicle is selected between 30 N to 36.8 N.

6. The kit as claimed in claim 1, wherein acidifying agent is selected from hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, trichloroacetic acid and mixtures thereof.

7. The kit as claimed in claim 1, wherein in the second solution concentration of colour developing agent in vehicle is between 0.4 to 1% w/v.

8. The kit as claimed in claim 1, wherein concentration of first solution acidifying agent in water is between 0.1 to 36.8 N solution.

9. A method for colour development for detection of Abscisic acid in urine sample as indication of livestock pregnancy using kit as claimed in claim 1, comprising of: Step 1—Collection of livestock urine sample in empty tubes, Step 2—Addition of 0.1 to 0.5 mL of first solution of acidifying agent using graduated dropper and mixing, Step 3—Addition of 0.2 to 0.5 mL of second solution of colour developing agent in vehicle using graduated dropper and mixing to develop the colour solution, Step 4—Comparing the developed colour with standard colour bands to detect the concentration of Abscisic acid in urine sample for indication of pregnancy in said livestock, wherein development of red colour indicates pregnancy and Yellowish orange colour indicates non pregnancy.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0046] Further aspects and advantages of the present invention will be readily understood from the following detailed description with reference to the accompanying figures of the drawings. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages but not limiting the scope of the invention. In the accompanying drawings:

[0047] FIG. 1: Illustrates an image of colour development a) before and b) after Interference removal from Urine sample upon acid pre-treatment as per present invention.

[0048] FIG. 2: Illustrates colour bands a) positive control for Abscisic acid about 10 μg/ml and above as indication of pregnancy and b) negative control for lower concentration of Abscisic acid as indication of non-pregnancy.

[0049] FIG. 3: Illustrate an image of colour development with respect to Effect of different acidifying agent on detection of pregnancy as per present invention kit.

DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention relates to a visual kit for pregnancy detection in livestock using urine sample, its composition and its method of use for Abscisic acid (ABA) level estimation in urine sample. The said Pregnancy detection kit comprises of a specific reagent composition A and B. To a specific amount of urine when Reagent A and B are added sequentially a visual colour change is instantaneously observed based on the amount of Abscisic acid present. A red colour in presence of Abscisic acid about 10 μg/ml and above as indication of pregnancy and Yellowish orange colour with lower amount of Abscisic acid as indication of non-pregnancy, which is then matched with the provided standard colour bands.

[0051] In the present invention, the kit for visual detection of livestock pregnancy status comprises Reagent A, Reagent B, glass/plastic transparent tubes and calibrated droppers and standard colour bands to match the colour generated in the samples. The method is rapid, based on simple visual detection, cost effective and can be performed at the livestock/animal side providing major advantage.

[0052] The reagent sample mentioned above is composed of two parts, A] an acidic composition of acids (Normality: 0.1N to 36.8N) such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, etc. and B] a reagent solution of ABA specific dye such as parahydroxy benzaldehyde (PHB) in sulfuric acid solution (0.4 to 1% w/v) (Normality: 30 to 36.8N).

[0053] Accordingly, the present invention relates to a kit for visual detection of livestock pregnancy in urine sample, which provides a practical and cost effective approach more specifically the advantages of portable reagent test kit assembly of present invention, coupled with its simple on site method of use make the invention economically viable with very high commercial application.

[0054] The present invention therefore provides kit for visual detection of livestock pregnancy status in urine samples on site. The said pregnancy detection kit offers an advantage such as simple portable kit, simple method of use, suitable combination of materials and tools, no withdrawal of blood sample, no biological sample preprocessing, rapid, visual colour based pregnancy estimation, affordable and thus has great outreach. The ease of handling and estimation is facilitated by kit design and its method of use based on minimum and cost effective materials and tools. This invention also offers a major advantage of minimal cost and early on site rapid visual pregnancy detection.

[0055] The present invention furthermore discloses a kit for visual detection of livestock pregnancy status in urine samples onsite which can overcome limitations of current estimation such as cost, travel, inconvenience and time. Detection can be done in farm settings where commonly used facilities for early, rapid, pregnancy detection is not available and also in hospitals, dispensaries, rural clinics, veterinary field. Thus, it provides a simple, economic yet effective solution to the existing lacuna.

[0056] Accordingly, kit for visual detection of livestock pregnancy status comprises, (i) Reagent A in a tube comprising of acidic composition of acids (Normality: 0.1 N to 36.8 N) such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid etc. (ii) Reagent B in tube comprising of ABA specific reagent parahydroxy benzaldehyde (PHB) in the concentration of 0.4 to 1% w/v in sulfuric acid (Normality: 30 N to 36.8 N), (iii) Empty tubes for collecting urine samples (iv) Calibrated Dropper for transferring Reagent A & Reagent B sequentially in sample tube (vi) Standard colour bands for visual comparison.

[0057] In accordance to second embodiment, the present invention provides a method to detect the pregnancy using said kit. The method of use of the kit for visual detection of livestock pregnancy status involves the following steps [0058] Step 1—Collection of livestock urine sample in empty tubes [0059] Step 2—Addition of specific amount of reagent [A] into sample tube with the help of provided calibrated dropper and mixing [0060] Step 3—Addition of reagent B into sample tube with the help of provided calibrated dropper and mixing [0061] Step 4—Compare the developed colour with standard colour bands.

[0062] Wherein, a red colour develops in presence of Abscisic acid about 10 μg/ml and above as indication of pregnancy and Yellowish orange colour develops at lower concentration of Abscisic acid as indication of non-pregnancy, which is then matched with the provided standard colour bands.

[0063] The foregoing description of the invention has been set merely to illustrate the current stage of invention and is not intended to be limiting. Since further development on detection techniques such as to record the reading in digital form/image form or introducing/collaborating the available techniques for minimizing the scope of errors or maximizing the detection and sensitivity of the disclosed embodiments is possible. Therefore, such modification will towards incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the disclosure.

[0064] The present invention is further illustrated in connection with particular examples as follows.

Example 1: Reaction of Abscisic Acid with Colour Developing Agent Parahydroxy Benzaladehyde (PHB)

[0065] A. Preparation of Abscisic acid (ABA) standards: Standard ABA was dissolved in distilled water and following concentrations were made. A=Blank, B=10 μg/mL, C=0.2 μg/mL and D=0.02 μg/mL.

[0066] B. Preparation of Para-hydroxy Benzaldehyde (PHB) Reagent (Second regent): About 100 mg of PHB was dissolved in 15 ml of Sulfuric acid (30 to 36.8 N)

[0067] Reaction: To the 0.4 ml of blank solution three ABA standard samples 0.6 ml of PHB reagent was added and observed for colour development.

[0068] Result: Increased concentration of ABA leads to development of red colour in the sample (Tube A-pinkish, Tube B-pinkish red/red, Tube C-Pink and Tube D-pinkish).

Example 2: Optimization of p-Hydroxybenzaldehyde (PHB) Concentration (i.e. Second Solution of Colour Developing Agent in a Vehicle)

[0069] The different concentrations of PHB was prepared in varied concentration of Sulfuric acid (H.sub.2SO.sub.4) (30 to 36.8 N). The concentration of PHB was ranging from 1 mg to 10 mg/0.6 mL (about 0.1 to 2% w/v) of Sulfuric acid (H.sub.2SO.sub.4) (30 to 36.8 N). Then 0.6 mL of this reagent was added to the 0.4 mL of standard ABA samples corresponding to Pregnant (ABA, 10 μg/mL) & Non-pregnant (ABA, 0.02m/mL) concentrations of ABA in cattle urine.

[0070] Result: It was found that 3 to 5 mg PHB in 0.6 mL of H.sub.2SO.sub.4 (30 to 36.8 N) showed a good colour discrimination between different ABA concentrations corresponding to low and high concentration of ABA. Therefore, decided to use this concentration of PHB 3 to 5 mg/0.6 ml of Sulfuric acid (30 to 36.8 N) to develop the kit of present invention.

Example 3: Experiments Using Cattle Urine Sample

[0071] A. Detection of Abscisic acid present in Cattle Urine: To the 0.4 mL of cattle urine samples (pregnant & non-pregnant), 0.6 mL of PHB in sulphuric acid (30 to 36.8 N) was added and observed for colour difference.

[0072] Result: Dark reddish brown colour was developed in all samples and difficult to distinguish between pregnant (+Control) and non-pregnant urine (−Control) samples as can be seen in FIG. 1a.

[0073] As cattle urine contains many components that might interfere with the PHB solution, there was need to remove such interfering substances.

[0074] B. Removal of Interference using acidifying agent pre-treatment (need of first solution): Urine samples were pre-treated with acidifying agent H.sub.2SO.sub.4 (0.1 to 36.8 N) to remove interference and observed better discrimination. These pre-treated urine samples were then reacted with PHB solution & were assessed for colour development in pregnant and non-pregnant cattle urine samples.

[0075] Result: A confirm visual colour difference was observed between pregnant (+Control) and non-pregnant (−Control) cattle urine (FIG. 1b). [0076] Reason for adding Acidifying agent in Reagent A (first solution): The acid addition removes interference associated with Urine samples and provides better colour discrimination between Pregnant and non-pregnant samples

[0077] Any Acid could be added as an Acidifying agent and can be selected from HCl, H.sub.2SO.sub.4, HNO.sub.3, Phosphoric acid, Acetic acid, Trichloroacetic acid and etc in the normality range of 0.1 to 36.8 N.

[0078] C. Optimization for Reagent A (first solution) and Reagent B (second solution) volumes: [0079] a) In first case, acidifying agent Reagent A—H.sub.2SO.sub.4 (0.1 to 36.8 N) was kept constant 0.4 mL and the amount of Reagent B [PHB in H.sub.2SO.sub.4; 4 mg/0.6 mL (30 to 36.8N)] was varied from 0.1 to 0.5 mL. Colour difference was analysed. [0080] b) In second case, the amount of Reagent B (PHB in H.sub.2SO.sub.4; 4 mg/0.6 mL) was kept constant as 0.3 mL and the amount of acidifying agent Reagent A (H.sub.2SO.sub.4; 0.1 to 36.8 N) was varied from 0.1 mL to 0.5 mL. Again, colour difference for final optimization was analysed.

[0081] Result: From both the variations, it was observed that at 0.1 to 0.5 mL of acidifying agent Reagent A H.sub.2SO.sub.4 (0.1 to 36.8 N) and 0.2 to 0.5 mL of Reagent B [PHB in H.sub.2SO.sub.4; 4 mg/0.6 mL (30 to 36.8 N)] showed good colour difference between pregnant and non-pregnant cattle urine. Hence, used these optimized amounts of reagents for point of care test kit preparation for pregnancy detection in cattle.

Example 4: A Performance of Pregnancy Test Kit as Per Present Invention

[0082] A. Method for using Kit and its Protocol: Pregnant and non-pregnant cattle urine samples were tested as per the kit procedure mentioned below.

[0083] To the 1 ml Cattle Urine sample added 0.2 ml of first solution Reagent A (sulfuric acid 0.1 to 36.8 N) followed by 0.3 ml of second solution Reagent B [PHB in H.sub.2SO.sub.4; 4 mg/0.6 mL (30 to 36.8 N)] and developed colour was observed and matched with standard color bands as shown in FIG. 2, red colour indicates pregnancy (FIG. 2a) and yellowish orange colour indicates non-pregnancy (FIG. 2b)

[0084] Result: The pregnant urine sample developed a red colour (+control) and non-pregnant urine sample developed a yellowish orange colour (−Control) as can see in FIG. 1b. The test procedure was distinguishing between both the samples.

Example 5: Effect of Different Acids as Vehicle for PHB Colour Developing Agent

[0085] Effect of different acids as PHB vehicles was evaluated. PHB was added 100 mg in 15 ml of all acids separately and mixed for 15 min. The reagent was observed for uniform mixing and appearance.

[0086] Result: Only concentrated acids dissolved PHB. The results are depicted in below Table 1.

TABLE-US-00001 TABLE 1 Effect of using different acids as vehicle for PHB reagent Acid PHB volume amount Name of Acid (ml) (mg) Observation Remark Conc HNO.sub.3 15 100 Dissolved Not suitable completely but for kit use generates Strong irritating fumes Conc HCl 15 100 Not dissolved Not suitable completely, for kit use generates strong irritating fumes Conc H.sub.2SO.sub.4 15 100 Dissolves PHB and Suitable for no fumes generation Kit use Phosphoric 15 100 PHB Not dissolved Not suitable acid completely Trichloroacetic 15 100 PHB Not dissolved Not Suitable acid completely Acetic acid 15 100 PHB Not dissolved Not suitable glacial completely [0087] Reason for adding PHB in Acid vehicle: PHB reacts with ABA and this reaction is acid catalysed. Acid dilution with water affects this reaction and loses interpretation thus normality of the acid must be at 30 to 36.8 N.

Example 6: Effect of Different Acidifying Agent: It can be Selected from any Acids HCl, HNO.SUB.3., H.SUB.2.SO.SUB.4., Phosphoric Acid, Acetic Acid, and Trichloroacetic Acid as Acidifying Agent in the Normality Range of 0.1 to 36.8 N

[0088] Wherein, 10% v/v of different acids were used as Reagent A first solution and added to 1 ml of cattle urine sample (pregnant & non pregnant).

[0089] Then Reagent B i.e. PHB in sulphuric acid H.sub.2SO.sub.4 (30 to 36.8 N) was added to develop a colour: The results are depicted in below FIG. 3 and Table 3. Any acid in the normality range of 0.1 to 36.8 N can provide discrimination between pregnant and non-pregnant cattle urine samples.

TABLE-US-00002 TABLE 2 Normality of different acids Normality of Normality 10% concentrated v/v diluted Name of Acid solution solution HNO.sub.3 15.8 1.58 HCl 11.65 1.16 H.sub.2SO.sub.4 36.8 3.68 Phosphoric acid 45.6 4.56 Trichloroacetic acid (20% w/v) 1.22 0.12 Acetic acid glacial 17.4 1.74

TABLE-US-00003 TABLE 3 Colour developed in samples with use of different acidifying agent. Colour with Urine Colour with Urine Acidifying agent sample of sample of non- (10% v/v) used Pregnant cattle Pregnant cattle H.sub.2SO.sub.4 Red Yellowish orange Acetic acid glacial Red Yellowish orange Phosphoric acid Red Yellowish orange Hydrochloric acid Red Yellowish orange

Example 7: Stability Data for Kit—the Stability of Reagent was Assessed for 3 Months at 2-8° C., 25° C., and 40° C. the Reagent was Evaluated for Discriminating Colour Development when Reacted with 1 ml of Pregnant and Non-Pregnant Cattle Urine Samples as Per the Kit Procedure

[0090] Result: Stability is depicted in Table 4 as correct colour development observed for both samples (i.e. a red colour in Pregnant cattle urine and yellowish orange colour in Non-Pregnant Urine samples). The reagent was found stable at all storage conditions and is able to discriminate between Pregnant and Non-Pregnant Urine samples.

TABLE-US-00004 TABLE 4 Stability of PHB reagent Period in Months 2 to 8° C. 25° C. 40° C. 1 month Stable Stable Stable 2 months Stable Stable Stable 3 months Stable Stable Stable