RU2703280C1 - Method of preparing samples of material for identification of nematode species by maldi toff biotyper method - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine and veterinary science and can be used for identification of species composition of nematodes by MALDI TOFF Biotyper method. That is ensured by mechanical ultrasonic homogenisation of the nematode head end, which is preliminarily treated with broad-spectrum antibiotics. After homogenisation, the biomaterial is placed in 0.9 % NaCl solution, centrifuged and the supernatant is withdrawn for further analysis by mass spectrometry. Mass-spectrometric proteomic profiles of the analyzed objects are obtained using the MALDI Sepsityper kit in the Flex control program and MALDI Biotyper with the "blood culture project" software and constructing graphs in Flex Analisis, allowing to perform species identification of Dirofilaria and Ascaris.EFFECT: disclosed is a method of preparing material samples for identifying a nematode species by the MALDI TOFF Biotyper method.1 cl, 1 tbl, 5 dwg, 4 ex

Description

The present invention relates to the field of medicine and veterinary medicine and relates to a method for preparing samples of parasitic pathogens, in this case, nematodes (Ascaris, Dirofilaria) for their identification using the MALDI TOFF Biotyper method (MALDI: matrix-activated laser desorption / ionization (TOFF - time of flight - analyzer flying time).

Proteomic studies (determination of a pathogen based on analysis of its protein spectrum) are a relatively new area of laboratory diagnosis of infectious diseases. There are no domestic analogues of the use of mass spectrometric profiles of nematodes for the diagnosis of parasitosis in the available literature.

The first studies of the potential of MALDI TOFF for studying the protein profile of helminths with an unknown genome were undertaken in 2007 by Brazilian experts [1]. Subsequently, this method was applied with the aim of differentiating protoscolex proteins of multichamber echinococcus [2]. Also, Korean specialists studied changes in the protein profile of cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory antigens [3]. In addition, studies were conducted on the differentiation of protein profiles of male and female individuals of the pathogen of Japanese schistosomiasis [4]. This method has shown its effectiveness as a method for the rapid diagnosis of nematodes parasitizing on crops [5].

The "gold standard" for the diagnosis of human dirofilariasis is the morphological identification of helminths removed during surgery. However, in case of damage to the nematode during its extraction, the diagnostic significance of this method is significantly reduced.

One of the important components of this method is the preparation of material samples for the study.

A known method of producing a somatic antigen D.immitis [6], in which helminths are placed in physiological solution in a ratio of 1:10, crushed in a mortar in the cold to obtain a homogeneous mass, the proteins are extracted for 24 hours in the same solution at 4 ° C on a magnetic stirrer, upon completion of extraction, the homogenate is centrifuged at 15,000 rpm for 30 minutes in the cold, the precipitate is removed, and the supernatant is fractionated by gel chromatography on a 16 × 70 cm column. In this method, sexually mature ones were used to obtain the antigen to helminths D.immitis, from dogs. The antigen obtained by this method was species-specific, but it had a rather high percentage of false-negative results (16%).

A known method for the identification of microorganisms using Bruker. This method allows the identification of pathogens of bacterial infectious processes. To conduct MALDI-TOFF MS (mass spectrometer) of blood and / or urine, a lysis buffer from the MALDI Sepsityper kit (Sepsitvper is the name of the test system) (Bruker Daltonics) is added to the biomaterial to remove the cells, then the washing buffer is carried out for additional purification additionally washing with 96% ethanol and bidistilled water. Further, to improve the quality of the spectrum, extraction is carried out in 70% formic acid, followed by layering of 50% acetonitrile. SNAs (α-Cyano-4-hydroxycirmamicacid) were applied as a matrix and placed in a mass spectrometer for analysis. Protein mass profiles of the studied objects are obtained using Microflex LT MALDI TOFF MS (Bruker Daltonics) with Flex Control software (Bruker Daltonics) and Maldi Biotyper, (Biotyper is the name of the device), visualized using Flex analysis 3.3 software (Bruker Daltonics). The method allows to obtain protein profiles of bacteria freed from other impurities, including blood cells [7].

However, to date, this method has not been used to obtain protein profiles of parasitic pathogens.

The closest technical solution to the proposed invention is a method for producing a purified somatic antigen Dirofilaria immitis. [8]. The presented method includes mechanical homogenization, centrifugation of the homogenate, selection of the supernatant and its use as an antigen. In this case, the head end is 2 cm long of the mature female Dirofilaria immitis, which is placed in a 0.25 M aqueous solution of sucrose in a ratio of 1: 3, frozen at a temperature of -18 ° C, mechanical homogenization and protein extraction are performed in a 0.25 M aqueous solution of sucrose at 4 ° C for 12 hours, then ultrasonic homogenization of the supernatant is carried out at 70 kHz 5 times for 30 sec und with an interval of 30 seconds in 3 cycles at 0 ° C, the supernatant formed after ultrasonic homogenization is dissolved in chilled acetone at a temperature of 0 ° C in a ratio of 1:20 with an exposure of 1 hour at a temperature of 4 ° C. The presented invention allows to obtain an antigen with high sensitivity and specificity and can be used in the diagnosis of dirofilariasis in humans and animals.

(See the patent of Russia No. 2525688, class IPC S07K 39/00; publ. 08.20.2014.)

The disadvantage of this method is that when conducting matrix-activated laser desorption / ionization (MALDI-TOFF MS) of biological material obtained as a result of this method of sample preparation, it was not possible to obtain clear and identical protein profiles of dirofilariae.

The aim of the invention is: to develop a method for sample preparation of nematodes for identifying the species composition by the MALDI TOFF Biotyper method with a high degree of specificity.

In the proposed method, only the cephalic end of sexually mature and immature nematodes of the genus Ascaris and Dirofilaria is used. Due to the fact that it is here that the glandular cells of the esophagus contain stenocytes (the least differentiated and common for each type of nematode).

The technical result of the invention is to obtain protein mass profiles of objects of study with high sensitivity and specificity, which can be used in the diagnosis of parasitic invasions of humans and animals.

The problem is solved in that the method of preparing material samples for species identification of nematodes using the MALDI TOFF Biotyper method, including mechanical, ultrasonic homogenization of an individual, centrifugation of a homogenate, selection of a supernatant for subsequent identification differs from prototypes in that: only the head end of the nematodes is used for homogenization; the biomaterial is pre-treated with broad-spectrum antibiotics; the biomaterial is preliminarily placed in 0.9% NaCl solution. In this case, clear mass spectrometric proteomic profiles are obtained using the MALDI Sepsityper kit in the Flex control program and MALDI TOFF Biotyper with the “blood culture project” software (the program for blood culture research) and graphing is performed using the Flex Analisis software product species identification of dirofilariae and ascaridate.

The proposed method is illustrated by the following materials.

In FIG. 1 is a table showing the ratio of reagents for preparing samples of biomaterial for the MALDI-TOFF Biotyper study.

In FIG. 2 is a graph illustrating example 1 (in the description) to the method according to the source [8]. Protein profiles of D.immitis (left) and D.repens (right). Punched in manual mode.

In FIG. 3 is a graph illustrating example 2 (in the description) to the method according to the source [7]. Protein profiles of D.immitis (left) and D.repens (right).

In FIG. 4 is a graph illustrating the result when using the patented sample preparation method. Protein profiles of D.immitis (left) and D.repens (right).

In FIG. 5 is a graph illustrating the result when using the patented sample preparation method. Protein profiles of A. Suum (left) and A. Lumbricoides (right).

The method includes the following steps:

Preparation of homogenized nematodes. An individual is washed many times in a 0.9% NaCl solution and placed for 24 hours in a 0.9% NaCl solution with the addition of 100 units / ml phenoxymethylpenicillin (penicillin V) and 100 μg / ml streptomycin. Separate the head end of the nematode 2 cm long and place it in a sterile 0.9% NaCl solution in a ratio of 1: 3 (parasite mass: volume of isotonic solution). The resulting mass is subjected to freezing at -18 ° C for 30 minutes. The mechanical homogenization of the frozen material is carried out in an ice bath until a liquid mass is obtained, followed by repeated freezing. This stage is performed 5 times. The resulting biomass is placed in tubes of the eppendorf type, which are placed in frozen 70% alcohol and ultrasonically homogenized at 70 kHz for 30 seconds five times.

200 μl of lysis buffer from the MALDI Sepsityper kit is added to the obtained biomass, mixed on a Vortex apparatus. Centrifuged for 2 minutes at 13,000 rpm. Remove supernatant. Suspend the precipitate in the wash buffer by pipetting. Centrifuge 2 minutes at 13,000 rpm. The precipitate is then suspended in 300 μl of deionized water and 900 μl of ethanol. In accordance with the table (see Fig. 1) add a certain amount of 70% formic acid and acetonitrile in a test tube and mix. 1 μl of the supernatant was collected and applied to the MALDI target point. After drying in air, the extract was coated with 1 μl of a solution of a matrix of CHSA (α-Cyano-4-hydroxycirmamicacid) and allowed to dry completely at room temperature.

Identification is carried out in the MALDI Biotyper device in accordance with the instructions for the device and the author's technique.

Example No. 1. Use during homogenization of dirofilariae 0.25 M aqueous solution of sucrose and 10 μl of formic acid and 10 μl of acetonitrile. The work was carried out in the Flex control program and MALDI Biotyper with the “normal project” software.

10 D.immitis and 10 D.repens were taken. The helminths were homogenized in a 0.25 M aqueous solution of sucrose in a ratio of 1: 3. They were frozen at -18 ° C, mechanical homogenization was performed 5 times from the frozen state in an ice bath, ultrasonic homogenization of the supernatant at 70 kHz 5 times for 30 seconds in five cycles at 0 C. 1 ml of biomass formed after ultrasonic homogenization was taken to eppendorf. For cell lysis, lysis buffer from the MALDI Sepsityper kit was added and vortexed for 10 seconds. Then centrifuged at 13000 rpm within 2 minutes. The supernatant was removed. The precipitate was suspended in the wash buffer by pipetting. Centrifuged again at 13,000 rpm. within 2 minutes. The supernatant was removed, 900 μl of 96% ethanol and 300 μl of bidistilled water were added. Vortex was shaken for 10 seconds. It was centrifuged at 13,000 rpm for 2 minutes. The supernatant was removed. To improve the quality of the spectrum, 10 μl of 70% formic acid was added to the obtained biomaterial (7.5 ± 2.5 μl) (Fig. 1 table). Then, 10 μl of 50% acetonitrile was added to the tube (see Table 1). Vortex was shaken for 10 seconds. The homogenate was centrifuged at 13,000 rpm for 2 minutes. One μ1 sample supernatant was applied to a steel plate (Bruker Daltonics) in two sequences. The target was dried for several minutes (5-15) at room temperature. Then, μ1 matrix of SNA (α-Cyano-4-hydroxycinnamicacid) was applied to each sample, dried and placed in a mass spectrometer for analysis. Homogenized protein mass profiles were obtained using Microflex LT MALDI-TOF MS (Bruker Daltonics) with FlexControl software (Bruker Daltonics) visualized using Flex analysis 3.3 software (Bruker Daltonics). The work was carried out in the Flex control program and MALDI BiotyperRTC with the “normal project” software. When plotting the graphs in Flex Analisis, they received a large number of fixed noises and fuzzy protein peaks. The construction of mass spectrometric profiles was carried out in manual mode. The differences between the species D.immitis and D.repens in the graphs could not be identified (Fig. 2).

Example No. 2. The use of isotonic sodium chloride solution, 20 μl of formic acid and 20 μl of acetonitrile during homogenization of dirofilariae. The work was carried out in the Flex control program and MALDI Biotyper with the “normal project” software.

5 D.immitis and 5 D.repens were taken. Helminths were homogenized in an isotonic sodium chloride solution in a ratio of 1: 3. They were frozen at -18 ° C, mechanical homogenization was performed 5 times from the frozen state in an ice bath, ultrasonic homogenization of the supernatant at 70 kHz 5 times for 30 seconds in five cycles at 0 ° C. The biomaterial formed after ultrasonic homogenization was taken 1 ml into eppendorf. For cell lysis, lysis buffer from the MALDI Sepsityper Kit-50 kit (Bruker Daltonics) was added and shaken on a vortex for 10 seconds. The precipitate in the wash buffer was suspended by pipetting. To improve the quality of the spectrum, 20 μl of extraction with 70% formic acid was added. Then, 20 μl of 50% acetonitrile was added to the tube (Fig. 1. Table). The homogenates were centrifuged at 13,000 rpm for 2 minutes. One μ1 sample supernatant was applied to a steel plate (Bruker Daltonics) in two sequences. The target was dried for several minutes (5-15) at room temperature. Then, μ1 matrix of SNA (α-Cyano-4-hydroxycinnamicacid) was applied to each sample, dried and placed in a mass spectrometer for analysis. Homogenized protein mass profiles were obtained using Microflex LT MALDI-TOF MS (Bruker Daltonics) with FlexControl software (Bruker Daltonics) visualized using Flex analysis 3.3 software (Bruker Daltonics). (fig. 3). The work was carried out with the “normal project” software. When plotting graphs in Flex Analisis, they received a small amount of recorded noise and fuzzy protein peaks. The differences between the species D.immitis and D.repens in the graphs could not be identified (Fig. 3).

Example No. 3. The use of isotonic sodium chloride solution, 20 μl of formic acid and 20 μl of acetonitrile during homogenization of dirofilariae. The work was carried out in the program Flex control and MALDI BiotyperRTC with the software “blood culture project)). When plotting graphs in Flex Analisis.

5 D.immitis and 5 D.repens were taken. Helminths were homogenized in an isotonic sodium chloride solution in a ratio of 1: 3. It was frozen at -18 ° C, mechanical homogenization was performed 5 times from the frozen state in an ice bath, ultrasonic homogenization of the supernatant at 70 kHz 5 times for 30 seconds in five cycles at 0 C. The biomaterial formed after ultrasonic homogenization was taken 1 ml into eppendorf . For cell lysis, lysis buffer from the MALDI Sepsityper Kit-50 kit (Bruker Daltonics) was added and shaken on a vortex for 10 seconds. The precipitate was suspended in the wash buffer by pipetting. To improve the quality of the spectrum, 20 μm extraction of 70% formic acid was added. Then, 20 μl of 50% acetonitrile was added to the tube (see table 1). The homogenates were centrifuged at 13,000 rpm for 2 minutes. One μ1 sample supernatant was applied to a steel plate (Bruker Daltonics) in two sequences. The target was dried for several minutes (5-15) at room temperature. Then, μ1 matrix of SNA (α-Cyano-4-hydroxycinnamicacid) was applied to each sample, dried and placed in a mass spectrometer for analysis. Homogenized protein mass profiles were obtained using Microflex LT MALDI-TOF MS (Bruker Daltonics) using FlexControl software (Bruker Daltonics) visualized using Flex analysis3.3 software (Bruker Daltonics) (Fig. 3). The work was carried out in the Flex control program and MALDI Biotyper RTC with the “blood culture project” software. When plotting in Flex Analisis. Due to the fact that an isotonic sodium chloride solution and another program were used, clear, comparable results were obtained (Fig. 4.)

The graph shows differences between D.immitis and D.repens.

Example No. 4. Use in ascaridate homogenization of isotonic sodium chloride solution, 20 μl of formic acid and 20 μl of acetonitrile. The work was carried out in the Flex control program and MALDI Biotyper RTC with the “blood culture project” software. When plotting in Flex Analisis.

10 young, immature roundworms were taken: 5 A. lumbricoides that had naturally departed in patients of the 5 A. suum clinic isolated from pig intestines in slaughterhouses. To carry out the MALDI-TOF MS analysis of helminths, they were washed in an isotonic sodium chloride solution with the addition of 100 units / ml phenoxymethylpenicillin (penicillin V) and 100 μg / ml streptomycin, homogenized mechanically and ultrasound. For cell lysis, lysis buffer from the MALDI Sepsityper Kit-50 kit (Bruker Daltonics) was added and shaken on a vortex for 10 seconds. The precipitate in the wash buffer was suspended by pipetting. To improve the quality of the spectrum, 20 μm extraction of 70% formic acid was added. Then, 20 μl of 50% acetonitrile was added to the tube (see table 1). The homogenates were centrifuged at 13,000 rpm for 2 minutes. One μ1 sample supernatant was applied to a steel plate (Bruker Daltonics) in two sequences. The target was dried for several minutes (5-15) at room temperature. Then, 1 μ1 CHCA matrix (α-Cyano-4-hydroxycinnamicacid) was applied to each sample, dried and placed in a mass spectrometer for analysis. Homogenized protein mass profiles were obtained using Microflex LT MALDI-TOF MS (Bruker Daltonics) with FlexControl software (Bruker Daltonics) visualized using Flex analysis3.3 software (Bruker Daltonics). The work was carried out in the Flex control program and MALDI BiotyperRTC with the “blood culture project” software. When plotting in Flex Analisis. Due to the fact that an isotonic sodium chloride solution was used with the addition of 100 units / ml phenoxymethylpenicillin (penicillin V) and 100 μg / ml streptomycin and another program, good results were obtained with other types of helminths. The presented invention allows to identify nematodes with a high degree of accuracy (Fig. 5).

List of references

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IR, Neves-Ferreira AG, Valente RH, Mota EM, Lenzi HL, Perales J. Improved protein identification efficiency by mass spectrometry using N-terminal chemical de-rivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome. J Mass Spectrom. 2007, 42 (6): 781-92.

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3. Pak JH, Moon JH, Hwang SJ, Cho SH, Seo SB, Kim TS. Proteomic analysis of differentially expressed proteins in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products. (J. Cell Biochem. 2009, 108 (6): 1376-88.

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8. Patent for invention No. 2525688 Method for producing purified somatic antigen Dirofilaria immitis, 06.23.2014 Nagorny S.A., Vaserin Yu.I. Krivorotova E.Yu.

Claims (1)

A method of preparing material samples for identifying the type of nematodes using the MALDI TOFF Biotyper method, including mechanical, ultrasonic homogenization of an individual, centrifugation of a homogenate, selection of a supernatant for subsequent identification, characterized in that the head end of the nematodes used for homogenization is pre-treated with broad-spectrum antibiotics and placed at 0 , 9% NaCl solution, while mass spectrometric proteomic profiles of the objects of study are obtained using the MALDI Sepsityper software package MME Flex control and MALDI Biotyper software «blood culture project» and perform charting Flex Analisis, allowing to carry out the identification of the species Dirofilaria and ascarids.

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