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Hybridization of nucleic acids. Nucleic acids

The specific characteristics of any organism depend on a specific sequence of nucleic acids contained in its genome.

The nucleic acids themselves are composed of block chains, each of which consists of sugar (deoxyribose or ribose, depending on whether the nucleic acid is DNA or RNA), a phosphorus-containing group and one of four organic purine or pyrimidine bases. DNA consists of two such chains arranged in a double helix and interconnected by bonds between organic bases. The bases selectively bind adenine with thymine and guanine with cytosine. It is the sequence of bases that makes organisms unique.

Development of probes (sample) nucleic acid-based

Nucleic acid probes are small segments of single-stranded nucleic acid that can be used to identify specific genetic sequences in samples. Probes (probes) can be designed for DNA or RNA sequences. The attractiveness of using gene probes in the determination of microorganisms is that the probe, consisting of a sequence of only 20 nucleotides, is unique and can be used to accurately determine the microorganism [52].

To detect the binding of the probe to the DNA or RNA of the microorganism being detected, it must be attached to some label that is easy to detect. At first, the work was carried out with radioisotope labels (for example, based on radioactive phosphorus P32), which could be determined by autoradiography or scintillation counting. The use and disposal of radioactive isotopes, however, is inevitably fraught with safety problems, which makes them unsuitable for standard tests in food production laboratories. Therefore, for the widespread use of nucleic acid probes, different labels were required.

A significant amount of research has been devoted to tagging probes using the Avidin-Biotin linkage. The basis of this technique is the high specificity of the relationship between avidin and biotin. The probe nucleic acid sequence is labeled with biotin and interacts with the detected DNA. Then, avidin bound to a suitable indicator, for example, avidin-alkaline phosphatase, is added, and binding is then detected by the formation of a colored product in a colorless substrate. The use of such new labeling systems showed that probes with non-radioactive tags can be used to detect microorganisms, but they are much less sensitive than systems with radioisotope tagging, and the increase in the number of cells (compared with the radioisotope system) required to determine reaches 100 .

To create non-isotopic probes with a sensitivity approaching that of isotopic labels, it was necessary to study alternative targets for probes in cells. DNA cell oriented probes attach to only a few regions on the chromosome of a cell of a designated microorganism. By studying the regions of cellular nucleic acid that are present in a relatively large number of copies in each cell and orienting the probes to these sites, the sensitivity of non-isotopic probes can be significantly increased. Work on increasing the sensitivity of probes has focused on the use of RNA as a target. RNA is a single-stranded nucleic acid that is present in cells in various forms. In one form, it is found in ribosomes, which are part of the protein synthesis system in the cell. Such RNA (known as ribosomal RNA - rRNA) is present in cells in a large number of copies. By orienting probes from nucleic acids to ribosomal RNA, the sensitivity of the analytical system can be significantly increased.

Samples of microorganisms in foods

Nucleic acid hybridization procedures for determining pathogenic bacteria in food products are described for Salmonella [35,47], Listeria [73,74], Yersinia enterocolitica [56,66], Listeria monocytogenes [38] producing Escherichia coli enterotoxins [55, 57, 87] ], producing enterotoxins of Staphylococcus aureus [91], Clostridiumperfringens and Clostridium botulinum [134].

The first commercially available analytical system for food products based on nucleic acid probes was introduced by Gene Trak Systems (Framingham, Massachusetts, USA) in 1985 [47]. It used probes that are selective for salmonella DNA and for chromosomal DNA-based determination of salmonella in fortified food samples. The analysis procedure included the hybridization of the detected DNA associated with the membrane filter and probes labeled with phosphorus 32. The total analysis time consisted of 40-44 hours of sample enrichment in non-selective and selective media and the subsequent hybridization procedure, which lasted 4-5 hours. Thus, the total analysis time was about 2 days. A test of the Salmonella test in the USA showed that it was at least equivalent to standard methods using cultures [49]. Gene Trak created a hybridization analysis system for Listeria [73] varieties based on this approach.

Gene Trak probe kits have been certified in the United States, and a number of laboratories have begun to use them. In Europe, however, food production laboratories were reluctant to use radioisotopes. In addition, phosphorus-32 has a short half-life, which caused difficulties in transporting the kits to remote areas. In 1988, Gene Trak began supplying non-isotope labeled probes for Salmonella, Listeria, and Escherichia coli with a colorimetric detection system. To overcome the decrease in sensitivity caused by the use of non-isotopic labels, the target nucleic acid in the cell was ribosomal RNA. The number of copies of this nucleic acid per cell is estimated at 500-20 LLC.

The colorimetric hybridization analysis is based on a hybridization reaction in liquid between the detected rRNA and two separate oligonucleotides using DNA probes (immobilized probe and signaling probe) specific for the microorganism being determined. The molecules of the immobilized probe using enzymes supplemented with a polymer consisting of approximately 100 residues of deoxyadenosine monophosphate. Molecules of the signaling probe chemically mark the hapten with fluorescein.

After appropriate enrichment of the test product, the sample is transferred to a test tube, and the microorganisms decompose, releasing the detected rRNA. Immobilized are then added, and signaling probes hybridize. If the detected rRNA is present in the sample, then hybridization occurs between the probes and the determined rRNA. The solution containing the probe-detectable microorganism complex is then brought into contact with a sensor containing the bound deoxythymidine homopolymer (under conditions under which hybridization between the polydeoxyoxyadenosine polymer of the immobilized probe and the polydeoxythymidine on the sensor is possible). Non-hybridized nucleic acids and cell debris are then washed away, leaving the immobilized DNA-RNA complex attached to the surface of the sensor. A linked fluorescein signaling probe is determined by adding an antifluorescein antibody conjugated to a peroxidase enzyme. Subsequent addition of a chromogenic substrate for the enzyme results in a color that can be measured with a spectrophotometer.

The results of colorimetric analyzes [88] showed a good agreement between the probe methods and the traditional culture-based methods for Salmonella and Listeria. It turned out that the sensitivity of the kits lies between 105 and 106 of the determined microorganisms per 1 ml, in connection with which the enrichment procedure is an important step. Following the introduction of these three kits, Gene Trak began producing systems for Staphylococcus aureus, Campylobacter and Yersinia enterocolitica.

Nucleic acid probes commercially available for confirming the presence of Campylobacter, Staphylococcus aureus, and Listeria are available from Gen probe (Gen Probe Inc., San Diego, USA). These kits are based on a single-stranded DNA probe complementary to the ribosomal RNA of the microorganism being detected. After the ribosomal RNA is isolated from the microorganism, the labeled DNA probe combines with it and forms a stable hybrid of complementary DNA and RNA. A hybridized probe can be detected due to luminescence.

A hybridization protection method based on the use of a chemiluminescent acridine ester is also used for analysis. This ether reacts with hydrogen peroxide under alkaline conditions, emitting light that can be measured in a luminometer. Acridine esters are covalently linked to synthetic DNA probes via an alkylamine chain. The analysis is based on differential chemical hydrolysis of the ether bond. The hydrolysis of this bond makes acridine permanently non-chemiluminescent. When the DNA probe to which the ester is attached hybridizes with the target RNA, acridine is protected from hydrolysis and may therefore become luminescent. Campylobacters Listeria assay kits use a lyophilized reagent probe. Campylobacter probe reacts with C. jejuni, C. coli and C. pylori; Listeria probe reacts with L. monocytogenes. In both cases, significant enrichment of crops is required before using the probe for confirmatory testing.

Evaluation of a set of probes for L. monocytogenes [21] showed that it is absolutely specific to the target organism. A positive result required the presence of about 106 L. monocytogenes. This kit seems to be a quick and reliable test to confirm the presence of culture and can be used directly with the broth for enrichment, thereby further reducing the duration of the test.

Future probes

The development and use of probes in the food industry has not progressed well in recent years. The released sets demonstrate their great potential, but are not used as widely as immunological tests. Microbiologists should always consider the usefulness of analyzing cellular genetic information. It is possible, however, that advances in molecular biology mean that the best way to obtain such information is through the use of nucleic acid amplification methods (for example, the polymerase chain reaction - CRP).

Methods of nucleic acid amplification

In recent years, several genetic amplification methods (amplification methods) have been developed and improved. These methods typically rely on the biochemical amplification of cellular nucleic acid and can lead to an increase in the number of copies per 2-3 h in 107 time. A very rapid increase in the number of targets achieved using nucleic acid amplification methods makes them ideal for developing systems for accelerated detection of microorganisms. Currently, a number of amplification methods have been developed used to determine microorganisms:

  •  polymerase chain reaction (CRP) and its variants, including nested CRP, reverse transcriptase CRP (RNA-dependent DNA polymerase) and multiple CRP;
  •  Q-beta replicase;
  •  ligase amplification reaction (LAR) \
  •  transcript amplification (TAS), also known as Self Sustained Sequence Replication (3SR) or nucleic acid sequence amplification (NASBA, Nucleic Acid Sequence Based Amplification).

Of these amplification methods, only CRP was introduced into the industry in the form of a technique based on a kit for determining food microorganisms. Various studies have been carried out using NASBA, there are a number of works describing the application of this method for the determination of foodborne pathogens, but there are still no commercially available analysis kits on the market.

Polymerase chain reaction (PCR)

CRP is a method used for repeated in vitro enzymatic synthesis of specific DNA sequences using two short oligonucleotide primers These primers hybridize to opposite strands of the DNA molecule and are adjacent to the region of interest of the target DNA. CRP flows through a series of secondary cycles, including DNA denaturation, annealing of primers, and an increase in primers under the action of DNA polymerase. These three stages of each cycle are controlled by changing the reaction temperature, since each stage only occurs at certain temperatures. These temperature changes are carried out using a special device for thermal cycling. The products of increasing primers after one cycle serve as templates for the next cycle, and thus the number of copies of the target DNA doubles in each cycle.

Chain reaction "reverse transcriptase polymerase" (CROT-P)

This reaction involves the use of a target RNA for CRP. CRP must run on a DNA molecule, and therefore, initially reverse transcriptase is used to obtain a copy of DNA, after which this copy is used in traditional CRP. The reverse transcriptase polymerase chain reaction (CROT-P) is particularly suitable for certain microbiological tests. Some food viruses contain RNA as the genetic material. Therefore, if amplification and, accordingly, the determination of viruses are necessary, it is necessary to use CROT-P. The second application of CROT-P is the identification of viable microorganisms. One of the problems associated with CRP is its high sensitivity and ability to increase very low concentrations of a detectable nucleic acid. Thus, when using CRP to determine the presence or absence of a specific microorganism in a food product, this reaction can determine the microorganism even if it was previously made inactive due to the corresponding technological process. This can lead to a false positive result. To overcome this problem, you can use CROT-P, aimed at informational RNA cells, which is produced only by active cells and after the formation has a short half-life. Thus, the determination of specific messenger RNA (infRNA) using CROT-P indicates the presence of viable microorganisms.

NASBA

NASBA is a multi-enzyme multiple amplification procedure that requires more enzymes and reagents than standard PCRs. Its advantage is that it is an isothermal procedure, and therefore, all stages of the reaction occur at the same temperature, which makes the device for thermal cycling unnecessary. A number of works have been published on the use of NASBA for the determination of foodborne pathogens (see, for example, [128]), but this procedure has not yet found wide application.

Industrial kits based on PCR

Currently, PCR-based kits for determining microorganisms in food products are produced by three firms. The IVC kit (Qualicon, USA) uses tablet reagents, a traditional thermal cycling device and a method based on gel electrophoresis. Samples with a positive reaction are visible as bands on the gel for electrophoresis. VAC kits are available for Salmonella [9], Listeria of the genus Listeria monocytogenes, and for E. coli 0157: Я7. Tests for Salmonella and E. coli 0157: #7 were tested and certified by the Association of the Research Institute of Chemical Analysts (AOACRI).

The second commercially available group of CRP kits is the Probelia kit (Sanofi, France), which uses a traditional CRP followed by immunological analysis and the Salmonella and Listeria colorimetric determination system.

The last available system for the central heating system is the TaqMan system (Perkin-Elmer, USA). It uses a new probe system, including the TaqMan Label tag.It does not fluoresce in its original form, but after the probe is connected between the CRP primers, it can be affected by the DNA polymerase enzyme used in the CRP to form the fluorescent final product. This fluorescence is determined using a special detection system. TaqMan kits for Salmonella are available, kits for Listeria and E. coli 0157 are being developed. One of the most interesting prospects for using TaqMan is quantification. Currently, all systems based on CRP are used to determine the presence or absence of microorganisms, and TaqMan methods and instruments can provide information on the actual number of microorganisms, that is, CRP is used to quickly calculate them.

Isolation of microorganisms from the food products and their concentration

In recent years, there has been significant interest in the potential for isolating microorganisms from food and then concentrating them to obtain a higher quantity per unit volume. This interest is caused by the fact that many existing methods of express testing have limited sensitivity - for example, 104/ Ml for ATP luminescence, 106/ ml - for electrical measurements, 105-106/ ml - for immunological analysis and DNA probes, about 103/ ml - for existing complete sets on the basis of CRP. These sensitivity levels mean that the growth period usually required before the rapid determination can significantly increase the total analysis time.

One way to solve this problem is to separate and concentrate microorganisms from food products so that microorganisms can be detected in higher concentrations. An additional advantage is that the cells of microorganisms can be separated from the food matrix, which in some cases may contain materials that interfere with the determination. A simple example of the use of concentration is the analysis of pure liquids (water, clear soft drinks, wines, beer, etc.), in which pollution levels are usually very low. Therefore, to concentrate microorganisms in a small area, large volumes of them are subjected to membrane filtration. Delayed microorganisms can then be analyzed. A detailed analysis of concentration methods is given in [11]. Five categories of such methods can be distinguished: filtration, centrifugation, phase separation, electrophoresis and immune methods.

Of these categories, only the immune methods have reached the stages of industrial production for use in the analysis of solid foods. The immunomagnetic compartment is based on the coating of small magnetic particles with specific antibodies for a specific cell. Coated particles can be added to a food suspension or culture medium, and in the presence of detectable cells, they attach to antibodies on these particles.

The use of a magnetic field holds particles with cells attached to them, allowing you to drain excess fluid and food residues, and thereby separate the cells from the food matrix and concentrate them. Such a system is produced by Dynal (Norway), LabM (Great Britain) and Denka (Japan), and Foss Electric (EIAFOSS) makes an automated system based on this procedure. Kits for Salmonella, Listeria, E. coli 0157, other verocytoxin-producing E. coli and Campylobacter are produced by different companies. Systems implementing immunomagnetic separation methods to determine the presence of E. coli 0157 are widely used, and in many countries these methods have become standard.

Detection and identification of microorganisms characteristics

After isolating microorganisms from a food product, it is sometimes necessary to establish which microorganism it is. This is especially important if it is supposed to be a pathogen. Traditionally, biochemical or immunological analyzes, as well as purified microorganisms, have been used in identification methods. The progress of molecular biology has made it possible to identify microorganisms by the structure of their DNA. The sensitivity of DNA-based methods actually makes identification possible to a level lower than the species (usually this procedure is called characterization or subtyping). Subtyping is a powerful new tool used by microbiologists not only to identify a microorganism, but also to establish its origin. Therefore, in some cases, it is possible to isolate the microorganism in the finished product, and then using a structured series of tests to find whether its source was a certain raw material, the environment in the production area or poorly washed parts of the equipment.

A number of DNA-based analysis methods have been developed that make subtyping possible (many of which are discussed in [17]), but only one method has been fully automated and has become available to microbiologists in food processing laboratories. This is ribotyping with a Qualicon RiboPrinter (Qualicon, USA). This purified automated bacterial colony is fed into this fully automated instrument and samples of DNA ranges (RiboPrint structures) are obtained, which are automatically compared with databases for identification and characterization. The method has been successfully applied in the food industry to determine pollution, identify sources and routes of pollution, as well as verify the authenticity of crops [12].

The future of microbiological methods

Traditional microbiological methods have not changed significantly in the last few decades. For the counting, detection and identification of microorganisms in samples, microbiologists mainly continue to use long-term enrichment and methods based on the growth of microorganisms on agar. With the development of food production technology, the need for ever faster microbiological data is constantly growing.

The accelerated development of the chilled products market and the production of products with relatively short shelf lives have led to the creation of accelerated automatic methods and systems for their implementation. Their use makes it possible: a) testing of raw materials before use; b) real-time monitoring of the sanitary-hygienic state of technological lines; and c) testing of finished products in a shorter time. All this should lead to the creation of higher quality products with extended shelf life.

All methods discussed in this chapter are currently used in food production laboratories. Some methods (for example, electrical) have been developed, implemented and used for quite some time, while others (for example, PCR) were developed much later. All these methods have good prospects, are accepted as standard and are not completely new. Some food manufacturers are already beginning to understand the benefits of combining different express methods to get even faster results. An example is the use of enzyme immunological analysis to detect the presence of Listeria subspecies, followed by the use of nucleic acid probes (specific for microorganism species) to confirm the presence or absence of L. monocytogenes.

One of the problems of some express methods is their lack of sensitivity. In many cases, this means that a long enrichment is required before using them. Research into methods for separating and concentrating microorganisms from food samples would allow microorganisms to be separated from food residues and concentrate them, eliminating the need for lengthy incubation procedures. Developments in the field of DNA methods for detection and identification / characterization lead to the creation of new tools for food production microbiologists. There is no doubt that in the future they will provide such opportunities for analysis that it is difficult to imagine today.

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