RU2562588C2 - Set for testing sperm in spots on analysed sample - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of forensic medical examination, namely to test sets, and makes it possible to carry out testing sperm in spots on analysed sample. Set contains two test strips of filter paper, containing: citrate buffer of 50 mmol/l concentration - 10 ml, α-naphthyl phosphate of 10 mmol/l concentration - 26.5 mg, fast red TR of 1.5 mmol/l concentration - 3.9 mg; and the other strip is soaked with solution, containing: sodium tartarate of 2 mmol/l concentration - 10 ml, α-naphthyl phosphate of 10 mmol/l concentration - 26.5 mg, fast red TR of 1.5 mmol/l concentration - 3.9 mg; distilled water for obtaining water extract of tested sample and reservoir for carrying out test. Sensitivity of strips to sperm constitutes not less than 0.00625 mg/ml.

EFFECT: invention makes it possible to accelerate and simplify process of analysis for presence of sperm in analysed sample both in laboratory conditions and directly on crime scene, as well as makes it possible to preserve analysis results.

2 dwg

Description

The invention relates to the field of forensic medical examination, namely to test sets, and allows testing of sperm in spots on the test sample.

Known kit "Phosphatesmo KM" for testing sperm in spots on the test sample, containing indicator paper specific for acid phosphatase - CF (Khaldi N., Miras A., Botti K., Benali L., Gromb S. Evaluation of three rapid detection methods for the forensic identification of seminal fluid in rape cases // J. Forensic Sci. - 2004. - Vol. 49, No. 4. - P. 749-753). With a positive reaction, violet staining of the paper is observed. The reaction is not a substitute for microscopic examination with live sperm.

The drawback of the kit: using indicator paper (test strip) included in the kit, you can identify only the total CF, which can be detected in the stains on the test samples due to the presence of sperm and saliva, vaginal discharge, blood, plant juice , as well as due to contamination with their bacterial enzymes.

Prostatic CF, which is found only in semen, is not detected with this kit.

Known test kit "Acid phosphatase kinetics" company "ABRIS +" containing citrate buffer, sodium tartrate, α-naphthyl phosphate and durable red TR. The kit is used in medicine for the separation of total and prostatic acid phosphatase for diagnostic purposes (Lifshits VM, Sidelnikova VI // Biochemical analyzes in the clinic. Medical Information Publishing House, Moscow, 2001, pp. 72-74). This set is accepted by the authors as a prototype.

The disadvantage of the set is the high consumption of expensive reagents, significant time and labor costs and, therefore, the inappropriateness of use for the purposes of forensic medical examination.

The technical result of the invention is the creation of a kit for testing sperm in spots on a test sample, which allows to significantly reduce the cost, speed up and simplify the process of testing sperm, to provide the ability to test sperm in spots on a test sample both in laboratory conditions and directly at the scene.

The specified technical result is achieved in that the kit for testing semen in spots on the test sample according to the invention contains two test strips of filter paper, one of which is impregnated with a solution containing:

concentration citrate buffer 50 mmol / L 10 ml α-naphthyl phosphate concentration of 10 mmol / l 26.5 mg durable red TR concentration of 1.5 mmol / l 3.9 mg

and the other strip is saturated with a solution containing:

sodium tartrate concentration of 2 mmol / l 10 ml α-naphthyl phosphate concentration of 10 mmol / l 26.5 mg durable red TR concentration of 1.5 mmol / l 3.9 mg

distilled water to obtain an aqueous extract of the test sample and the capacity for testing, while the sensitivity of the strips to sperm is at least 0.00625 mg / ml.

Studies have shown that test strips of filter paper, impregnated with reagents from the well-known set of “Acidic Phosphatase Kinetics”, can significantly reduce the cost of expensive reagents. One set of reagents allows you to conduct about 2500 test studies. A single sperm test study (separation of total and prostatic acid phosphatase) takes about 30 minutes. The kit allows you to conduct research at the scene. Test strips accumulate biomaterial (sperm) on themselves during the reaction. They are dried, stored and subsequently, if necessary, used for other purposes of the forensic medical examination (DNA research, detection of antigens of the ABO system).

The invention is illustrated in FIG. 1, 2 where

in FIG. 1 shows test strips 1 (citrate buffer), 2 (sodium tartrate), container 4 for testing, a container with sterile distilled water 5;

in FIG. 2 shows an object from the surface of which fragments were cut for the test sample 3.

Test strips 1 and 2 included in the kit were prepared using reagents from the Acid Phosphatase Kinetics kit of ABRIS +. The composition of the kit includes the following ingredients: citrate buffer in an amount of 50 ml (concentration of 50 mmol / l); sodium tartrate in an amount of 50 ml (concentration of 2 mmol / l); α-naphthyl phosphate in an amount of 10 × 26.5 mg (concentration of 10 mmol / l); durable red TR in an amount of 10 × 3.9 mg (concentration 1.5 mmol / L).

Working reagent No. 1 is obtained by dissolving the contents of one bottle of α-naphthyl phosphate in 10 ml of citrate buffer. Transfer the resulting solution to one of the vials with strong red TR and mix until completely dissolved. Working reagent No. 1 is used to impregnate the test strip 1 with citrate buffer to detect total CF.

Working reagent No. 2 is obtained by dissolving the contents of one bottle of α-naphthyl phosphate in 10 ml of sodium tartrate solution. Transfer the resulting solution to a new vial with a solid red TR and mix until completely dissolved. Working reagent No. 2 is used to impregnate the test strip with 2 sodium tartrate to detect prostatic CF.

Filter paper tapes (0.3 cm wide and 9-15 cm long) are immersed in containers containing, respectively, working reagent No. 1 or working reagent No. 2, for at least 20 seconds to uniformly soak with the appropriate buffer. The impregnated filter paper tapes are dried in a thermostat at a temperature of 56 ° C for 30-40 minutes. Dried filter paper tapes are cut into test strips 1, 2 (reagent No. 1 and reagent No. 2) 0.3 cm wide and 1.5 cm long. The thus prepared test strips 1, 2 retain their properties for at least 48 hours.

The kit is used as follows.

Prepare a sample for research.

A fragment or several fragments 0.5 × 0.5 cm in size are cut from the surface of the object of study and used as the test sample 3. Fragments of the test sample 3 are placed in a container 4 and filled with the minimum required volume of distilled water 5. Extracted at a temperature of 4 ° C from 18 to 24 hours in a laboratory, or within 30 minutes - 1 hour at a temperature of 15-20 ° C, if work is carried out directly at the scene.

Sperm testing in the prepared extract of the test sample is divided into 2 stages.

Stage 1. General CF detection.

In the prepared aqueous extract (capacity 4) of the test sample 3, test strip 1 with reagent No. 1 is placed so that the upper part of the strip 1 protrudes above the liquid. 15-30 minutes after immersion of the strip 1 in the extract of the test sample 3 with a positive result, the protruding end of the test strip 1, and then the entire strip 1 is painted red-brown. With a negative result, the color of the strip is not observed.

2 stage. Detection of prostatic CF.

The second stage is carried out upon receipt of a positive test result in stage 1. Test strip 2 with reagent No. 2 is placed in the prepared aqueous extract of the test sample and the reaction proceeding in the tank 4 is observed. If no coloring of test strip 2 occurs within 15-30 minutes, the result is considered positive. This is explained by the fact that prostatic CF is blocked by tartrate, and after its suppression by an inhibitor (tartrate), dyeing of test strip 2 does not occur. If the result is negative, test strip 2 turns brownish. Testing is indicative and is not a substitute for microscopic examination with live sperm.

Studies have shown that test strips 1.2 with a size of 1.5 cm × 0.3 cm, when used successively, provide a positive test result with a concentration of sperm in the aqueous extract of the test sample of at least 0.00625 mg / ml.

The proposed kit for indicative testing of sperm in spots on the test sample is notable for its low cost and ease of use, it allows you to save the results of the study, which can be further used for DNA isolation and typing, as well as for detection of ABO system antigens by standard serological reactions.

Claims (1)

A set for testing semen in spots on the test sample, characterized in that it contains two test strips of filter paper, one of which is impregnated with a solution containing:
concentration citrate buffer 50 mmol / L 10 ml α-naphthyl phosphate concentration of 10 mmol / l 26.5 mg durable red TR concentration of 1.5 mmol / l 3.9 mg
and the other strip is saturated with a solution containing:
sodium tartrate concentration of 2 mmol / l 10 ml α-naphthyl phosphate concentration of 10 mmol / l 26.5 mg durable red TR concentration of 1.5 mmol / l 3.9 mg
distilled water to obtain an aqueous extract of the test sample and the capacity for testing, while the sensitivity of the strips to sperm is at least 0.00625 mg / ml.

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