RU2846880C1 - Mycobacterium avium rapid detection and identification test system - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a test system for detecting Mycobacterium avium. Said test system includes primers specific to the sequence of the insertion element IS1311 and containing nucleotide sequences SEQ ID NO: 1 and SEQ ID NO: 2, as well as a probe containing the nucleotide sequence SEQ ID NO: 3. Invention also relates to a kit of reagents for detecting Mycobacterium avium, comprising said test system and a reaction mixture, as well as a method for detecting Mycobacterium avium.

EFFECT: present invention provides detection of Mycobacterium avium mycobacteriosis agent DNA in the biomaterial by real-time PCR with specific primers and a fluorescent-labelled probe when using as a target multicopy gene IS1311, found only in Mycobacterium avium.

5 cl, 2 dwg, 5 tbl, 3 ex

Description

Field of technology

The invention relates to medicine and molecular biology. A test system is disclosed for detecting DNA of the mycobacterium avium pathogen in biological material using real-time PCR with specifically selected primers and a fluorescently labeled probe, using the multicopy IS1311 gene as a target. The invention can be used to create medical devices for in vitro diagnostics, to detect the mycobacterium pathogen by clinical laboratory diagnostic services in healthcare facilities, and to conduct epidemiological surveillance of the population to monitor the spread of mycobacterium pathogens.

State of the art

There is a steady increase in the proportion of mycobacteriosis in the structure of secondary diseases in HIV infection. In Russia, there is no centralized monitoring of this disease, however, according to some researchers, the prevalence of mycobacteriosis is 1.5-4.3 per 100,000 population (Beloborodova E.N. et al. Clinical and epidemiological characteristics of mycobacteriosis in two constituent entities of the Russian Federation // Infectious diseases. - 2020. - Vol. 18. - No. 1. - Pp. 35-42.). Risk groups for developing the disease are immunocompromised patients: persons with acquired immunodeficiency syndrome and other immunodeficiencies; after internal organ transplantation; patients with thermal injuries; patients with pneumoconiosis; patients with chronic obstructive pulmonary disease; patients with bronchial asthma; patients with cystic fibrosis; patients with bronchiectasis; patients with sarcoidosis; patients with pulmonary fibrosis of various origins; patients with oncological diseases, including hemoblastoses; individuals receiving long-term glucocorticosteroid or immunosuppressive therapy; the elderly. In addition, after COVID-19, many patients develop an immunodeficiency state and pronounced residual changes in the form of fibrosis develop in the lungs, which creates the prerequisites for the development of mycobacteriosis, the causative agent of which is widespread in nature (Yang F., Liu N., Hu J.Y. et al. Pulmonary rehabilitation guidelines in the principle of 4S for patients infected with 2019 novel coronavirus (2019-nCoV) (In Chinese)., Zhon-ghuaJie He He Hu Xi ZaZhi. 2020; 43 (3): 180-182.).

MAC infection is a true opportunistic AIDS-defining disease and develops in severe immunodeficiency in 20-40% of cases (more often with a decrease in CD4+ lymphocytes to less than 50 cells/μl) (Horsburgh CR Jr. The pathophysiology of disseminated Mycobacterium avium complex disease in AIDS. J Infect Dis 1999, Suppl 3: S461-465.; Nightingale SD1, Byrd LT, Southern PM, Jockusch JD, Cal SX, Wynne BA. Incidence of Mycobacterium avium -intracellulare complex bacteremia in human immunodeficiency virus-positive patients. J Infect Dis. 1992 Jun;165(6):1082-5) Mycobacterium avium is detected in more than 80% of such patients (Savchenko MA Clinical and Epidemiological aspects of mycobacteriosis in patients with HIV infection // HIV infection and immunosuppression. - 2019. - Vol. 11. - No. 2. - P. 27-33.).

Mycobacteriosis often mimics tuberculosis in its symptoms, but treatment for these patients varies greatly, as Mycobacterium avium is naturally resistant to many anti-tuberculosis drugs. To ensure a correct diagnosis and timely and appropriate treatment, rapid laboratory testing for Mycobacterium avium is essential.

Diagnosis of mycobacteriosis is carried out by culturing samples from patients on solid and liquid nutrient media, and to this day this method remains the “gold standard” for laboratory diagnosis of mycobacterial infections.

However, Mycobacterium avium are slow-growing microorganisms; therefore, even when using automated liquid media cultivation systems (BACTEC MGIT, etc.), the culture growth time may vary up to 42 days. In addition, after obtaining mycobacterial growth, differentiation from Mycobacterium tuberculosis complex and identification of Mycobacterium avium by other methods is required: biochemical; time-of-flight mass spectrometry (MALDI-ToF MS); molecular genetic (multilocus sequencing, restriction fragment length polymorphism (RFLP) analysis, 16S rRNA gene sequencing); high-performance liquid chromatography of mycolic acids (HPLC). The disadvantage of this approach is the duration of the research, since mycobacterial culture growth is required (up to 2-3 months), labor intensity and the need for expensive equipment, as well as the high cost of research. Molecular diagnostic methods based on the polymerase chain reaction (PCR) can reduce the time it takes to diagnose mycobacterial infections to just a few hours. Therefore, the development and implementation of new, effective and rapid laboratory diagnostic methods for mycobacteriosis is extremely important and relevant.

Known molecular methods and corresponding test systems (reagent kits) for the diagnosis of mycobacteriosis are based on the detection of specific DNA fragments in the genome.

The commercial GenoType Mycobacterium CM kit (Hain Lifescience GmbH) is based on PCR followed by hybridization on strips with immobilized oligonucleotide probes and differentiates the most common species of non-tuberculous mycobacteria, including M. avium . However, this method is expensive and labor-intensive, cannot be automated, and has low sensitivity. Furthermore, according to the official instructions for the kit, only mycobacterial cultures are suitable for testing, which increases the time to obtain results to 2-3 months and limits the applicability of this method.

A number of patents concerning the detection of M. avium complex by molecular methods have been found in the foreign literature. They use hybridization with oligonucleotide probes (US5153119, 1992; US5314801, 1994; US6136529, 2000), strand displacement isothermal amplification (SDA) (US5681705, 1997; US5691143, 1997) and PCR (US5667994, 1997; US6465638, 2002). However, these test systems can only identify the MAC complex without differentiating to M. avium, which is critical for immunocompromised patients.

A method for detecting Mycobacterium avium using PCR is known (US8084212, 2011). The IS1245 sequence proposed by the authors as a target for M. avium is not present in all subspecies of this species (Johansen TB et al. Distribution of IS1311 and IS1245 in Mycobacterium avium subspecies revisited // Journal of Clinical Microbiology. - 2005. - Vol. 43. - No. 5. - Pp. 2500-2502).

The prior art also discloses a kit and method for detecting Mycobacterium avium (US2020181690), including the primers 5'-AGTGGGCAACAATCCAAGAG-3' and 5'-CCCGACACAACGAGGTTT-3. The method utilizes PCR followed by melting curve construction. The target in this case is a region of the gene encoding cytochrome P450 ( BJP76_RS07310 ). The reaction time is 180 minutes. However, this method utilizes the intercalating dye SYBR-Green I for detection. A weakness of intercalating dyes is their nonspecificity. If the wrong DNA region, or even several different regions, are amplified during PCR, an amplification curve is still constructed that is identical to the curve for amplification of the target genes. Intercalating dyes generate a fluorescent signal upon binding to any double-stranded DNA, regardless of its nucleotide sequence. This means that additional analyses are required to confirm the presence of specific amplification products. These types of analyses require highly skilled operators.

A method is known for identifying the species of mycobacteria of the MAIS complex ( M. avium , M. intracellulare , M. scrofulaceum ) and M. tuberculosis , including the stages of DNA extraction, restriction, separation of restriction products in agarose gel, followed by analysis of the restriction profiles of the studied species. DNA is extracted by heating mycobacteria for 12 min at 95°C in a TE buffer pH 8.0, the resulting amplicons are treated with Hin6I restriction enzyme (the volume of amplicons for restriction is 17 μl and the restriction time is 1 hour 10 min (RU2319748, 2008). The analysis consists of 3 stages:

1. The DNA sequence of the gene being studied is cloned using the PCR method.

2. The resulting amplicons are subjected to the action of restriction enzymes, which are selected using special programs, taking into account that the fragments into which the resulting amplicons are cut must be detected on an agarose gel.

3. Analysis of profile polymorphism after separation of restriction products in agarose gel.

However, this method uses PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism), which is more labor- and time-consuming, less sensitive, and carries a higher risk of contamination than real-time PCR. Analysis time is 110 minutes.

Known are oligonucleotide primers complementary to the Mycobacterium avium- specific region of the mig gene, used to detect Mycobacterium avium DNA, having the following nucleotide composition: 5'-CGT CAA AAG CGA ACT GCA -3' and 5'-TAA TTC GTT GCC CGA CTC -3', and a method for detecting Mycobacterium avium DNA using the above oligonucleotide primers by the polymerase chain reaction (PCR), including DNA extraction, amplification of DNA on oligonucleotide primers, transfer of the amplification product to a gel, followed by detection of the analysis results on a transilluminator, characterized in that the primers have the nucleotide sequences: 5'-CGT CAA AAG CGA ACT GCA -3' and 5'-TAA TTC GTT GCC CGA CTC -3' in the case of a positive reaction A fragment corresponding to a size of 157 bp is synthesized (RU2554842, 2015). DNA electrophoresis requires specialized training, as after the amplification reaction, the tubes (strips, plates) containing the amplicons must be opened and placed into the wells of an agarose or polyacrylamide gel along with the dye. Furthermore, gel preparation takes an average of 20 minutes, and the electrophoresis process itself takes 20 minutes (depending on the length of the PCR product). This increases the reaction time by at least 40 minutes. Consequently, the use of electrophoretic detection after PCR increases the time and labor costs, reduces sensitivity, introduces contamination into the laboratory, and is less sensitive.

Thus, currently available methods for detecting M. avium include PCR with strip hybridization, PCR-RFLP, and PCR with electrophoretic detection. All of these methods require opening the tubes after the amplification step, which increases the risk of in-laboratory contamination and, consequently, false-positive results.

The technical problem solved by the claimed invention is the need to overcome the shortcomings inherent in analogues and the prototype by creating a test system for detecting DNA of the causative agent of mycobacteriosis Mycobacterium avium in biomaterial by the real-time PCR method with specifically selected primers and a fluorescently labeled probe, using as a target the multi-copy gene IS1311, found only in M. avium .

Disclosure of invention

The technical result of the proposed invention is to improve the quality of laboratory diagnostics of mycobacteriosis, i.e. the development of a method for the rapid detection of DNA of M. avium, the causative agent of mycobacteriosis (no more than 121 minutes), characterized by a reduced likelihood of intra-laboratory contamination and, accordingly, the likelihood of obtaining false positive results, as well as high specificity for M. avium .

The specified technical result is achieved by a test system for detecting Mycobacterium avium, including primers specific to the sequence of the insertion element IS1311 and a probe, where

- primers are oligonucleotides containing the nucleotide sequence SEQ ID NO: 1 - SEQ ID NO: 2,

- the probe contains the nucleotide sequence SEQ ID NO: 3.

The 5' end of the probe is labeled with the reporter fluorescent dye ROX, and the 3' end of the probe is labeled with the quencher dye BHQ-2.

The technical result is also achieved by a reagent kit for detecting Mycobacterium avium , comprising the claimed test system and a reaction mixture comprising: 5.1 mM MgCl ₂ ; 0.39 mM of each nucleoside triphosphate - dATP, dGTP, dCTP and dTTP; 0.05 U/μl Taq DNA polymerase; 1x PCR buffer. The PCR buffer used is of the following composition: 0.1 M Tris-HCl with pH = 8.6; 0.5 M KCl; 1% Tween20.

The technical result is also achieved by a method for detecting Mycobacterium avium, which consists of conducting real-time PCR using the claimed test system or a kit based on it.

Brief description of the drawings

The invention is illustrated by the following drawings.

Fig. 1 shows a diagram of the distribution of types of biomaterials among 318 clinical samples for testing the clinical characteristics of the developed test system.

Fig. 2 shows the DNA melting curves of the test samples obtained by real-time PCR using the developed test system. Both channels are presented on a single graph: FAM (BKO) (black curves) and ROX (MAC) (gray curves).

Implementation of the invention

A more detailed description of the claimed invention is provided below. The present invention is subject to various changes and modifications that would be apparent to those skilled in the art based on this description. Such changes do not limit the scope of the claims.

The developed test system uses 96-well plates with a volume of 0.2 µl containing lyophilized reagents for the amplification of DNA fragments specific to M. avium . One tube is used per sample, ensuring high throughput: 96-well amplifiers can analyze 94 samples in a single run (two additional wells include negative and positive controls). The reaction mixture contains oligonucleotides for the detection of an internal control sample added during DNA extraction, thus avoiding false negative results.

The reaction mixture contained: 5.1 mM _MgCl2 ; 0.39 mM of each nucleoside triphosphate (dATP, dGTP, dCTP, and dTTP); 0.05 U/μl Taq DNA polymerase; 1x PCR buffer (0.1 M Tris-HCl pH 8.6; 0.5 M KCl; 1% Tween20).

In addition, the mixture includes specific primers for the multi-copy sequence of the insertion element specific for M. avium, IS1311, at 0.45 μM:

SEQ ID NO: 1 5’-CGCTATCTCTCCGAGACCAC (IS1311_F5)

SEQ ID NO: 2 5’-TCTGCGAGTTTCCACTATGC (IS1311_R5)

As well as a probe including the sequence SEQ ID NO:3 labeled with the fluorophore ROX 5’-(ROX)ATTCAGCCGACCAATCCGTTGCC (BHQ2), at a concentration of 0.3 μM.

SEQ ID NO: 3 5'-(ROX)ATTCAGCCGACCAATCCGTTGCC (BHQ2)

The 5' end of the probe is labeled with the reporter fluorescent dye ROX, and the 3' end of the probe is labeled with the quencher dye BHQ-2.

Primers and probes were selected using Unipro UGENE software (Novosibirsk, Russia). A preliminary assessment of oligonucleotide characteristics for subsequent thermal cycling program selection was performed using the software's built-in "PCR in silico" function. Primer and probe annealing specificity was assessed using BLAST software (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Oligonucleotide synthesis was performed using standard technology commissioned by Vector-Best JSC (Novosibirsk, Russia).

When performing real-time PCR for MAC detection, a positive negative control (PNC and K-) is required. The positive control sample is a 1076-bp amplicon obtained by amplifying Mycobacterium avium DNA using the Mav_F1seq (5'-TCGGCCGCGCACACTG) and Mav_R1seq (5'-TCGCCTGAGGCTCTGC) primers.

The proposed test system for detecting M. avium uses the IS1311 insertion element (multi-copy target), which is found only in M. avium , as a target, which allows for a significant increase in the specificity of the reaction (due to the selection of a specific insertion element), as well as a decrease in the threshold for detecting the pathogen, i.e., an increase in the sensitivity of the reaction (due to the multi-copy target).

The amplification program was as follows: 1 cycle of 50°C for 2 min, 95°C for 2 min, then 50 cycles of 98°C for 10 sec and 60°C for 1 min with fluorescence signal detection in the FAM and ROX channels (total time to obtain the result: 112 minutes). The presence of a signal in the FAM channel indicates successful DNA extraction. A signal in the ROX channel, within the cutoff value (Ct < 40), indicates the presence of M. avium DNA. To assess the progress of the PCR reaction, positive and negative controls are required (a negative control of the PCR reaction (C-) and a negative control of the experiment that has passed the extraction stage (NCE)).

After completion of the amplification reaction, in the native software of the device for plotting melting graphs and obtaining the Ct value, the Treshhold line value is set equal to the average of the maximum value of the melting curve flare-up (“plateau”), divided by 40.

The interpretation of the results is carried out in 2 stages:

1. Sample validation: The shape of the obtained melting curves is assessed; it should be sigmoid (if the curve shape does not correspond to sigmoid, such samples are sent for re-sampling). Next, the sample extraction stage is assessed and invalid samples are rejected according to the following algorithm:

- Calculate the average Ct value for the FAM channel for all analyzed samples (including the LOQ and OQ). Next, calculate the difference between the obtained threshold value (Ct) and the average Ct value for the FAM channel (ΔCt). If the difference is more than two cycles from the average value, such samples are rejected and re-extracted and analyzed.

2. Interpretation of results according to the following algorithm:

- the sample is considered positive (containing Mycobacterium avium complex DNA) if the Ct in the “ROX” channel is less than or equal to 40;

A sample is considered negative (not containing Mycobacterium avium complex DNA) if the Ct value for the "ROX" channel is greater than 40 or undetectable. However, if the ΔCt ICD value for such a sample exceeds 2, the result is not considered negative. A repeat analysis of the sample is required, beginning with the DNA extraction step.

If a signal is observed in one or both channels of the K-channel that does not exceed the cutoff value, then contamination of the entire run is considered. Similarly, if a signal is observed in the "ROX" channel of the negative control (NCC) that does not exceed the cutoff value of 40, then contamination is considered. In the event of contamination, all positive results for this PCR run are considered invalid. Positive samples must be retested. Samples that test negative are considered negative.

If the positive control sample of the reaction has no signal or is greater than the limit (0) on one or both channels, the setup is considered invalid and all samples must be re-assayed, after first ensuring that the expiration dates of the reagents have not been violated.

The invention is illustrated by the following examples.

Evaluation of analytical specificity and sensitivity

Based on the results of cultural studies, 81 clinical isolates of mycobacteria were selected and studied; the species composition is presented in Table 1.

Table 1. Species composition of the studied NTM population.

Types of NTM Number of isolates, abs. M. abscessus 3 M. asiaticum 1 M. avium 51 M. chelonae 1 M. chimaera-intracellulare 9 M. fortuitum 4 M. gordonae 1 M. kansasii 5 M. kumamotonense 1 M. malmoense 1 M. paragordonae 1 M. poriferae 1 M. timonense 1 M. xenopi 1

For these cultures, the mycobacterial species was determined using MALDI-TOF mass spectrometry and sequencing of the 16S rRNA, hsp65, rpoB, IS1311 genes and the 16S-23S intergenic spacer. RT-PCR was performed on a CFX96 instrument (Biorad, USA). The amplification program was as follows: 1 cycle of 50°C for 2 min, 95°C for 2 min, then 50 cycles of 98°C for 10 sec and 60°C for 1 min with fluorescence signal detection in the FAM and ROX channels (the total time to obtain the result was 112 minutes). The presence of a signal in the FAM channel indicates successful DNA extraction. Whereas, the presence of a signal in the ROX channel, within the limit value (Ct <40), indicates the presence of M. avium DNA. To assess the progress of the PCR reaction, positive and negative controls were used (negative control of the PCR reaction (K-) and negative control of the experiment that passed through the isolation stage (NEC)).

After completion of the amplification reaction, in the native software of the device for plotting melting graphs and obtaining the Ct value, the Treshhold line value was set equal to the average of the maximum value of the melting curve flare-up (“plateau”), divided by 40.

The results were interpreted in two stages:

1. Sample validation: The shape of the obtained curves corresponded to a sigmoid.) Next, the sample selection stage was evaluated and invalid samples were rejected according to the following algorithm:

The average Ct value was calculated for the FAM channel for all analyzed samples (including the LOQ and OQ). The difference between the obtained threshold value (Ct) and the average Ct value for the FAM channel (ΔCt) was then calculated. If the difference was more than two cycles from the average value, the samples were rejected and subject to re-extraction and reprocessing.

2. The interpretation itself was carried out as follows:

- The sample was considered positive (containing Mycobacterium avium complex DNA) if the Ct in the “ROX” channel was less than or equal to 40;

A sample was considered negative (not containing Mycobacterium avium complex DNA) if the Ct value for the "ROX" channel was greater than 40 or undetectable. However, if the ΔCt ICD value for such a sample exceeded 2, the result was not considered negative. In this case, the sample was reanalyzed, starting with the DNA extraction step.

If a signal in one or both channels of the K-channel was observed that did not exceed the cutoff values, contamination was detected. Similarly, if a signal in the negative control (NCC) channel of the "ROX" channel was observed that did not exceed the cutoff value of 40, contamination was detected. In the event of contamination, all positive results for this PCR run were considered invalid. In this case, positive samples were re-analyzed. Samples that tested negative were considered negative.

If the positive control sample of the reaction had no signal or was greater than the cutoff (0) on one or both channels, the setup was considered invalid and all samples were rearranged, after first ensuring that the expiration dates of the reagents had not been violated.

Analytical performance was assessed using the freely available calculator https://epitools.ausvet.com.au/testevaluation, based on sequencing results. Sensitivity was 96.8% [86.54–99.52], and specificity was 100% [88.43–100]. The results are presented in Table 2.

Table 2. Results of the evaluation of the analytical characteristics of the developed test system.

Detection of M. avium DNA sequencing Sensitivity Specificity PCR Discovered Not found Discovered 49 0 96.8%
[86.54-99.52] 100%
[88.43-100] Not found 2 30

Example 2

Evaluation of the detection limit of the developed test system

The detection limit was assessed using isolated M. avium DNA (50 μl), the concentration of which was measured using a DropDX-2044HT droplet digital PCR (ddPCR) system (RainSure Scientific, China). The reaction was carried out according to the standard droplet digital PCR protocol: 1 μl of a 20x primer and probe mixture (included in the developed test system) and 9 μl of DNA at various concentrations were added to 10 μl of PCR Mastermix.

The obtained results were calculated using the formula:

Table 3. Estimation of the detection limit of the developed test system

McFarland concentration Concentration determined by ddPCR copies/µl number of repetitions % positive Ct FAM, avg. CV,% Ct ROX, avg. CV,% 1 2.2x10 ⁷ 4 100 30.62 0.09 18.38 0.52 0.1 2.1x10 ⁶ 4 100 33.71 1.17 21.86 0.31 0.01 2x10 ⁵ 4 100 31.26 0.19 24.22 0.41 0.001 3.1x10 ⁴ 4 100 30.52 1.06 26.90 0.84 0.0001 6x10 ³ 4 100 30.23 0.10 29.95 0.58 0.00001 0 8 100 34.49 5.42 36.79 2.46 0.000001 n/a 24 100 34.86 5.68 39.59 2.70 0.0000001 n/a 24 75 30.77 0.30 18.38 0.52 0.00000001 n/a 24 0 30.62 0.09 0.00 0.00

Thus, the developed test system showed a detection limit of 10 ^-6 McFarland, which is equivalent to 23 CFU/ml, and in 75% of cases it allowed the detection of samples with a concentration of 2.3 CFU/ml.

Example 3

Evaluation of the clinical characteristics of the developed test system

Clinical characteristics were assessed using 318 clinical specimens from various types of biomaterials obtained during routine clinical trials at the National Medical Research Center for Physiological Research of the Ministry of Health of the Russian Federation. Respiratory lavage (BAL) and sputum samples constituted the majority (270 samples, 86.0%). The distribution of biomaterial types is shown in Fig. 1. The species diversity of the studied isolates is presented in Table 4.

Table 4. Species diversity of 318 clinical samples for testing the clinical characteristics of the developed test system.

Types of NTM Amount of NTM, abs. M. abscessus 21 M. asiaticum 1 M. avium 198 M. canariasense/cosmeticum 1 M. chimaera-intracellulare 25 M. farcinica 1 M. fortuitum 8 M. gordonae 6 M. kansasii 18 M. lentiflavum 16 M. malmoense 5 M. marseillense 2 M. paragordoniae 1 M. saskatchewanense 1 M. scrofulaceum 2 M. septicum 1 M. smegmatis 3 M. stephanolepidis 1 M. szulgai 2 M. xenopi 5

For these samples, the mycobacterial species was determined using MALDI-TOF mass spectrometry and sequencing of the 16S rRNA, hsp65 , rpoB , IS1311 genes and the 16S-23S intergenic spacer. RT-PCR was performed on a CFX96 instrument (Biorad, USA). The amplification program was as follows: 1 cycle of 50°C for 2 min, 95°C for 2 min, then 50 cycles of 98°C for 10 sec and 60°C for 1 min with fluorescence signal detection in the FAM and ROX channels (the total time to obtain the result was 112 minutes). The presence of a signal in the FAM channel indicated successful DNA extraction. Whereas, the presence of a signal in the ROX channel, within the cutoff value (Ct <40), indicated the presence of M. avium DNA. To assess the progress of the PCR reaction, positive and negative controls were used (negative control of the PCR reaction (K-) and negative control of the experiment that passed through the isolation stage (NEC)).

After completion of the amplification reaction, in the native software of the device for plotting melting graphs and obtaining the Ct value, the Treshhold line value was set equal to the average of the maximum value of the melting curve flare-up (“plateau”), divided by 40.

The results were interpreted in two stages:

1. Sample validation: The shape of the obtained melting curves was assessed; it should be sigmoid (if the curve shape did not correspond to sigmoid, such samples were sent for re-sampling). Next, the sample extraction stage was assessed, and invalid samples were rejected using the following algorithm:

The average Ct value was calculated for the FAM channel for all analyzed samples (including the LOQ and OQ). The difference between the obtained threshold value (Ct) and the average Ct value for the FAM channel (ΔCt) was then calculated. If the difference was more than two cycles from the average value, such samples were rejected and subject to re-extraction and analysis.

2. The results were interpreted according to the following algorithm:

- A sample was considered positive (containing Mycobacterium avium complex DNA) if the Ct in the “ROX” channel was less than or equal to 40.

A sample was considered negative (not containing Mycobacterium avium complex DNA) if the Ct value for the "ROX" channel was greater than 40 or undetectable. However, if the ΔCt ICD value for such a sample exceeded 2, the result was not considered negative. The sample was reanalyzed, starting with the DNA extraction step.

If a signal in one or both channels was observed in the K channel and did not exceed the cutoff values, contamination was detected. Similarly, if a signal in the negative control (NCC) channel "ROX" did not exceed the cutoff value of 40, contamination was detected. In the event of contamination, all positive results for this PCR run were considered invalid. Positive samples were reanalyzed. Samples that tested negative were considered negative.

If the positive control sample of the reaction had no signal or was greater than the cutoff (0) on one or both channels, the setup was considered invalid and all samples were rearranged, after first ensuring that the expiration dates of the reagents had not been violated.

Clinical characteristics were assessed using the freely available calculator https://epitools.ausvet.com.au/testevaluation , relative to sequencing results. Clinical sensitivity was 83.84% [77.96–88.68] and specificity was 94.17% [88.35–97.62]. The results are presented in Table 5.

Table 5. Results of the evaluation of the clinical characteristics of the developed test system.

Detection of M. avium DNA sequencing Sensitivity Specificity PCR Discovered Not found Discovered 166 7 83.84%
[77.96-88.68] 94.17%
[88.35-97.62] Not found 32 113

All tests described in the three examples were performed on a CFX96 real-time thermocycler from Bio-Rad (USA). An example of the resulting curves is shown in Fig. 2.

The M. avium detection test system we developed demonstrates high specificity and sensitivity, which contributes to the increased effectiveness of mycobacteriosis diagnostics. Furthermore, signal detection occurs without opening the tube (strip, plate) after the amplification stage, significantly reducing the risk of in-laboratory contamination. It also reduces the time and labor required to obtain the final result, as detection is performed in real time.

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<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..23</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q13">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>attcagccgaccaatccgttgcc</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="4">

<INSDSeq>

<INSDSeq_length>16</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..16</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q14">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tcggccgcgcacactg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="5">

<INSDSeq>

<INSDSeq_length>16</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..16</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q15">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tcgcctgaggctctgc</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (7)

1. A test system for the detection of Mycobacterium avium , including primers specific to the sequence of the insertion element IS1311 and a probe, where (1) the primers are oligonucleotides containing the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, (2) the probe contains the nucleotide sequence SEQ ID NO: 3. 2. The test system according to claim 1, characterized in that the 5'-end of the probe is labeled with the reporter fluorescent dye ROX, and the 3'-end of the probe is labeled with the quencher dye BHQ-2. 3. A reagent kit for detecting Mycobacterium avium , comprising a test system according to item 1 and a reaction mixture comprising: 5.1 mM _MgCl2 ; 0.39 mM of each nucleoside triphosphate - dATP, dGTP, dCTP and dTTP; 0.05 U/μl Taq DNA polymerase; 1x PCR buffer. 4. The kit according to item 3, characterized in that a PCR buffer of the following composition is used: 0.1 M Tris-HCl with pH = 8.6; 0.5 M KCl; 1% Tween20. 5. Detection methodMycobacterium avium, characterized by the fact that What real-time PCR is performed using the test system according to paragraph 1 or the kit according to paragraph 3.