RU2551958C1 - SET OF OLIGONUCLEOTIDE PRIMERS AND FLUORESCENT-LABELLED PROBES FOR IDENTIFICATION AND SUBTYPING OF BACTERIAL DNA OF Pasteurella multocida OF SEROTYPES A, B, D, E, F BY PCR METHOD IN REAL TIME - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a set of oligonucleotide primers and probes for the identification and subtyping of a bacterial DNA of Pasteurella multocida of serotypes A, B, D, E, F by the PCR method in real time. The invention can be used in genetic engineering and veterinary practice for the detection of a genetic material (DNA) of the bacteria P. multocida of the said serotypes.

EFFECT: improvement of the method.

2 dwg, 5 tbl, 2 ex

Description

The invention relates to sets of oligonucleotide primers and probes and can be used in biotechnology, in particular in genetic engineering, and veterinary practice to identify the genetic material (DNA) of the bacterium Pasteurella multocida serotypes A, B, D, E, F in clinical or sectional samples for clarification of the diagnosis, as well as for solving research problems in studying the properties of P.multocida bacteria, in assessing the effectiveness of diagnostic, prophylactic or therapeutic drugs against cattle pasteurellosis. The use of specific primers and probes makes it possible to identify the genetic material of P. multocida bacteria in the studied samples and to carry out typing of P. multocida bacteria by the method of polymerase chain reaction (PCR) with hybridization-fluorescence detection in real time.

Pasteurellosis of cattle (Latin, English - Pasteurellosis; pulmonary pasteurellosis, hemorrhagic septicemia, transport fever, enzootic bronchopneumonia of cattle) is a contagious infectious disease characterized by acute course of septic phenomena, croupous pneumonia, pleurisy, various pleurisy body, and with subacute and chronic currents purulent-necrotizing pneumonia. The causative agent of pasteurellosis - Pasteurella multocida - is a polymorphic, often short gram-negative, motionless ellipsoidal bacilli, located in isolation, in pairs or less often in chains, do not form a spore; aerobes and optional anaerobes. Pasteurella multocida is a bacterium that causes infectious diseases in many species of mammals and birds (Arumugam et al. 2011, Sharma et al. 2010; Shayegh et al. 2009; Varshney et al. 2009; Wilkie I.W. et al., 2012).

The severity of clinical manifestations and pathological changes during pasteurellosis are not the same and depend on many factors (feed or transport stress, bacterial or viral infections, fluctuations in the ambient temperature), as well as on the serovariant of the strain that caused the disease. Currently, all P.multocida strains circulating in nature are divided into five capsular serogroups, characterized on the basis of the antigenic characteristics of capsular polysaccharides, denoted by the Latin letters A, B, D, E and F and 16, somatic serotypes differing in lipopolysaccharide antigens ( 1 to 16) (Carter GR, 1961; Rimler RB, Rhoades KR, 1987).

These pathogens cause the greatest damage in beef and dairy cattle breeding, being important etiological agents of mass respiratory diseases of young animals. P. multocida causes outbreaks of respiratory diseases characterized by both acute manifestation and severe course, and with a tendency to subacute and chronic course.

Diagnosis of pasteurellosis is based on a complex of epizootological, clinical, pathological and laboratory studies. Morphological (microscopy of blood smears and smears from affected organs) and phenotypic (isolation of a pure culture on nutrient media with identification by biochemical properties) diagnostic methods, as well as determination of P. multocida serovariants, are used as the main laboratory methods for the primary identification of the pathogen. For species identification, determination of serotypes and serogroups of P. multocida, serological typing with specific rabbit antisera is performed. However, in our country, serological typing has not been developed.

As an alternative to serological typing, in the last decade for the diagnosis and typing of P.multocida, a method based on polymerase chain reaction (PCR) has been used. This method has several advantages, since it increases the accuracy of the species identification of bacteria, reduces the time for their detection, makes it possible to detect and type the pathogen in the test material without isolating a pure culture. In addition, various modifications of PCR make it possible to analyze in multiplex format.

An analogue is the PCR test system for detecting Pasteurella multocida (LSI VetMAX ™ Pasteurella multocida - toxinogenic strains - Real-Time PCR Kit manufactured by LSI, France; Pasteurella Multocida Real time PCR Kit, manufactured by Bio SB performs R&D, etc.) as well as laboratory variants of PCR test systems for detecting the bacteria Pasteurella multocida [Rocke TE,.,., Shadduck DJ 2002. A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1 // http://www.ncbi.nlm.nih.gov/pubmed/12061646 "\ o" Avian diseases., 46, 370-377.] . However, commercial variants of the PCR test systems do not determine the serotype of the bacteria, and the laboratory version of the PCR test systems for detecting the bacterium Pasteurella multocida, proposed by Rocke T.E. et al. in 2002, identifies only the 1st serotype of the bacterium.

     Known analogues are PCR test systems for determining the serogroups A, B, D, E and F of the bacterium Pasteurella multocida [Arumugam, ND, Ajam, N., Blackall, PJ, Asiah, NM, Ramlan, M., Maria, J. , Yuslan, S. and Thong, KL 2011. Capsular serotyping of Pasteurella multocida from various animal hosts - a comparison of phenotypic and genotypic methods // Trop. Biomed., 28, 55-63.

Ferreira T. S. P., Felizardo M. R., de Gobbi D. D. S., Gomes C. R., Filsner P. H. L. N., Moreno M., Paix˜ao R., Pereira J., Moreno A. M. 2012. Virulence Genes and Antimicrobial Resistance Profiles of Pasteurella multocida Strains Isolated from Rabbits in Brazil // Sci. World J., Vol. 2012, Article ID 685028, 6 pages].

     The closest analogue (prototype) is a PCR test system for determining the serogroups A, B, D, E and F of the bacteria Pasteurella multocida [Swaraj Rajkhowa, Ingudam Shakuntala, Seema Rani Pegu, Rajib Kumar Das, Anubrata Das. 2012. Detection of Pasteurella multocida isolates from local pigs of India by polymerase chain reaction and their antibiogram // Trop. Anim. Health Prod., 44, 1497-1503].

       However, the above test systems (analogues and prototype) have an electrophoretic mode of recording the result, which significantly increases the analysis time, because additional manipulations are required related to the DNA trituration of the test sample or amplicons obtained during PCR, which complicate the formulation of the analysis and can lead to false positive results. The electrophoresis stage leads to a sharp decrease in the likelihood of contamination of the studied samples with amplification products.

The technical result of the claimed invention is the development of a more sensitive set of specific oligonucleotide primers and probes for identification of the genetic material of P. multocida with the possibility of determining the serotype in biological fluids, environmental samples and other samples by real-time PCR using hybridization-fluorescence detection in real time.

The indicated technical result is achieved by a set of oligonucleotide primers and fluorescently-labeled probes for the identification and subtyping of the DNA of the bacterium Pasteurella multocida serotypes A, B, D, E, F by real-time PCR described in the application.

Real-time PCR does not require additional manipulations related to the DNA sampling of the test sample or amplicons obtained during PCR, which complicate the formulation of the analysis and can lead to false positive results. Such an approach allows us to abandon the stage of electrophoresis, which leads to a sharp decrease in the likelihood of contamination of the studied samples with amplification products, and also reduces the requirements for PCR laboratory.

The technical result was obtained by constructing diagnostic primers and fluorescently-labeled probes on a conservative site (cap locus) of the bacterial genome and optimizing the concentrations of the components of the reaction mixture and the conditions for PCR. A pair of primers and probes were designed to identify the genetic material of the bacterium P. multocida. To control amplification, recombinant plasmids were obtained that carried sections of the DNA template specific for each serotype.

At the initial stage, the analysis of the nucleotide sequences of the genomes of serotypes A, B, D, E, F of the bacterium P. multocida from the NCBI database (http://www.ncbi.nlm.nih.gov/) was performed and the most conserved regions were determined.

The analysis of the properties of oligonucleotide primers and probes was carried out using the Vector NTI 9.0.0 (InforMax) program.

To identify the genetic material of the bacterium P. multocida (Kmt1 gene) by real-time PCR, common primers and probe were selected, which are presented in table 1.

Table 1. The structure of the primers and probe for the detection of the genetic material of the bacterium P. multocida in the plot of the Kmt1 gene

Primers F 266-292 5-ATAAGAAACGTAACTCAACATGGAAATA-3
R 477-456 5-GAGTGGGCTTGTCGGTAGTCTT-3 Probe Z 322-346 5 (FAM) -AAACCGGCAAATAACAATAAGCTGA-3 (BHQ1)

To determine the serotypes A, B, D, E, F, P. multocida bacteria using real-time multiplex PCR, primers and probes were selected, the structure is shown in Table 2.

Table 2. The structure of primers and probes for detection of serotypes A, B, D, E, F bacteria P. multocida

Bacterium
(Gene target) Names of Primers and Probes Sequence (5 '→ 3') T annealing (° C) Amplicon Size (bp) P. multocida serogroup A
(cap locus) F9165-9187 5-TTCGTTAAAAATGACAGCTATGC-3 51,2 223 R9388-9367 5-ATAATCGTCAGAAGCTCATGCG-3 53.7 Z9217-9244 5 (R6G) -ATTTCTCAGCATTAACACATGATTGGAT-3 (BHQ1) 57.4 P. multocida serogroup B
(cap locus) F12541-12564 5-GCGTGTATAACCTACATCTTCCCA-3 54.1 167 R12708-12687 5-CGTCCATCAACACCTTTACTGC-3 53.8 Z12618-12643 5 (FAM) -TAGGCACAGAATATTCAAAACCCCGT-3 (BHQ1) 59.9 P. multocida serogroup D
(cap locus) F3306-3328 5-ATCGCATCCAGAATAGCAAACTC-3 54,2 355 R3661-3643 5-TCCGATGCTTTGGTTGTGC-3 54,2 Z3350-3379 (Cy5) -CCGATTAAACTCAAATCTAGGGACATACTT- (BHQ2) 58.0 P. multocida serogroup E
(cap locus) F4539-4556 5-TGGGCACATGCTCGCTTA-3 53.3 357 R4896-4872 5-CTGCTTGATTTTGTCTTTCTCCTAA-3 53.3 Z4631-4654 5 (ROX) -ATGTGGCAAAGCGATCAATTCAGA-3 (BHQ2) 59.5 P. multocida serogroup F
(cap locus) F2885-2905 5-CGGAGAACGCAGAAATCAGAA-3 54.5 257 R3142-3120 5-CAACAACGACTTCAAATGGGTAG-3 53.1 Z2947-2970 5 (R6G) -CTTGCTCCATTGCCAGATCATGTT-3 (BHQ1) 59.0

Example 1. Verification of the analytical sensitivity of a set of primers and probes.

By the method of molecular transformation of competent bacterial cells of Escherichia coli with plasmids pCR 2.1 containing specific DNA inserts of the bacterium P. multocida, positive control samples (FFP) were obtained to control amplification.

For real-time PCR, recombinant plasmid DNA was used as the analyzed samples, including a DNA insert corresponding to the detected genome regions of each serotype of the bacterium P. multocida.

To confirm the specificity of the obtained DNA fragments, their nucleotide sequence was determined; for this, the BigDye ^® Terminator v3.1 Cycle Sequencing Kits reagent kit (Applied Biosystems, USA) was used. Sequencing reaction products were analyzed by capillary electrophoresis in an automated sequencer ^{ABI PRISM ® 3130xl (Applied Biosystems /} Hitachi, Japan). The obtained nucleotide sequences were compared with the sequences of the NCBI BLAST database (http://www.ncbi.nlm.nih.gov/). All DNA fragments necessary for obtaining FFP were targeted and corresponded to parts of the bacterial genome of P. multocida.

The amplification conditions were optimized by the following parameters: the concentration of magnesium ions in the reaction mixture; the concentration of primers and probes in the reaction mixture; primer annealing temperature.

The optimized composition of the reaction mixture included the following components: 10 × Taq buffer without Mg ²⁺ (Medigen Laboratory, Russia) - final concentration 1 ×; 100 mM MgCl2 solution - final concentration 3.3 mM; 5 mM dNTP solution — final concentration of each 0.2 mM; primer mixture — final concentration of each 0.15 µM; probe mixture — final concentration of each 0.2 µM; SmartTaq DNA polymerase — final concentration 1.5 ea / μl; water for PCR. The total volume of the reaction mixture was 30 μl.

The analytical sensitivity of the method for each analyte was determined by setting up real-time PCR, where 10-fold dilutions of positive control samples were used as test samples (PKO / Pm-Kmt1; PKO / Mh; PKO / Pm-A, PKO / Pm-D , FFP / Pm-B, FFP / Pm-E and FFP / Pm-F), the characteristics of which are given in table 3.

Table 3. Characterization of positive control samples used to determine analytical sensitivity

FFP The length of plasmid DNA (bp) Concentration
(mcg / ml) The number of copies of DNA (GE / ml) FFP / P.m.-Kmt1 4142 2.40 5.3 × 10 ¹¹ FFP / M.h. 4057 2.99 6.8 × 10 ¹¹ FFP / P.m.-A 4157 1.10 2.4 × 10 ¹¹ FFP / P.m.-B 4098 1.34 3.0 × 10 ¹¹ FFP / P.m.-D 4286 2.25 4.8 × 10 ¹¹ FFP / P.m.-E 3979 1.19 2.8 × 10 ¹¹ FFP / P.m.-F 3982 0.85 2.0 × 10 ¹¹

Plasmid DNA concentration was determined using the Quant-iT dsDNA, HS reagent kit (Invitrogen, USA) and QUBIT fluorimeter (Invitrogen, USA).

Recalculation of DNA concentration in the number of copies was made in the converter program http://molbiol.ru/scripts/01_07.html according to the formulas:

m [g] = Q [mol] x Mw ^olig [kDa] x 10 ³

m [g] = Q [mol] x <Mw> [Da] x L [kb] x 10 ³

N [pieces] = Q [mol] x N _A

c [M] x V [L] = Q [mol],

where: m [g] is the weight of the nucleic acid;

Mw ^olig [kDa] is the molecular weight of the oligonucleotide.

<Mw> [Da] is the average molecular weight of one / base pair;

base = 324.5 Da

base pair = 649 Da

ribo base = 340.5 Da

Q [mol] is the amount of nucleic acid;

N [copies] is the number of nucleic acid molecules;

c [M] is the molar concentration of the nucleic acid;

V [L] is the volume in which the nucleic acid is dissolved;

L [kb] is the length of the nucleic acid;

N _A - Avogadro number = 6.022045x1023 [1 / mol].

The following is proposed for multiplex PCR analysis.

reaction setup format using 2-reaction mixtures to detect the following analytes:

Mix No. 1:

P.m.-Kmt1 - Pasteurella multocida (Kmt1 gene);

P.m.-A - Pasteurella multocida serogroup A;

P.m.-D - Pasteurella multocida serogroup D.

Mix No. 2:

P.m.-B - Pasteurella multocida serogroup B;

P.m.-E - Pasteurella multocida serogroup E;

P.m.-F - Pasteurella multocida serogroup F.

The results of determining the analytical sensitivity are given in

 FIG. 1 and 2.

FIG. 1 - assessment of the analytical sensitivity of the set of primers and probes

for mixture No. 1. Amplification and recording of results was carried out in the device

Rotor Gene 6000 (Corbett Research, Australia):

A - fluorescence curves on the FAM / Green channel of samples of PKO / P.m.-Kmt1;

B - fluorescence curves on the R6G / Yellow channel of samples of PKO / P.m.-A;

B - fluorescence curves on the Cy5 / Red channel of PKO / P.m.-D samples.

FIG. 2 - assessment of the analytical sensitivity of the set of primers and probes

for mixture No. 2. Amplification and recording of results was carried out in the device

Rotor Gene 6000 (Corbett Research, Australia):

A - fluorescence curves on the FAM / Green channel of samples of FFP / P.m.-B;

B - fluorescence curves on the ROX / Orange channel of PKO / P.m.-E samples;

B - fluorescence curves on the R6G / Yellow channel of PKO / P.m.-F samples.

For analytical sensitivity, the last dilution of FFP was taken, with which the result of PCR analysis was interpreted as positive. Samples with a Ct value not exceeding 40 were considered positive. The results of the experiments conducted to evaluate the analytical sensitivity of the method are shown in table 4.

Table 4. The results of determining the analytical sensitivity of a set of primers and probes for the detection of the bacterium P. multocida and its serotypes

FFP Analytical
sensitivity
(GE in the reaction) Ct value in the last
breeding detectable
positively FFP / P.m.-Kmt1 1.6 × 10 ² 37.83 FFP / P.m.-A 7.2 × 10 38.29 FFP / P.m.-B 9.0 × 10 35.38 FFP / P.m.-D 1.5 × 10 ³ 35.55 FFP / P.m.-E 8.1 × 10 39.28 FFP / P.m.-F 5.9 × 10 ² 35,99

The minimum amount of DNA matrices detected using our primers and probes after optimizing the reaction conditions, expressed in GE (genomic equivalents) in 30 μl of the reaction mixture, ranged from 72 to 1500 GE per reaction for different serotypes of P. multocida bacteria and 160 GE to the reaction for the bacterium P. multocida (Kmt1 gene).

Example 2. Determination and subtyping of P. multocida in samples.

The procedure for DNA extraction from the studied material was carried out using the reagent kit “Kit for the isolation of DNA / RNA from serum or blood plasma” (LLC NPF Litekh, Russia) in accordance with the instructions for use.

Real-time PCR for both determining P. multocida DNA and determining the subtype of bacteria was carried out in the reaction mixture of the following composition (for 1 study):

cDNA 5 μl 10 × Taq buffer without Mg ²⁺ 3 μl 100 mM MgCl ₂ solution 1 μl 5 mM dNTP solution 1 μl A mixture of primers and probes (2 p.u. each) 1 μl each Smart Taq DNA Polymerase 5 u / μl 0.3 μl Water for PCR up to 30 μl of total volume

Real-time PCR, both to determine the genetic material of P. multocida and to determine its subtype, and the results were recorded in a Rotor Gene 6000 instrument (Corbett Research, Australia) according to the following program:

Temperature (° C) Time (minutes: seconds) The number of cycles 95 05:00 one 95 00:10 45 54 00:20 72 00:20

The measurement of fluorescence was carried out at a temperature of 54 C.

For the study, P. multocida bacteria were washed from the nutrient medium (Hottinger agar with horse serum) and dried cultures of P. multocida bacteria subtypes diluted in physiological saline to a concentration of 1000 microbial cells / reaction. As negative controls, dried cultures of bacteria Mannheimia haemolytica type A1 (strain 16, a collection of microorganisms of the Institute of Experimental Veterinary Medicine of Siberia and the Far East of the Russian Academy of Agricultural Sciences) and Escherichia coli (strain F-50, ATCC 25922, a collection of microorganisms of the State Scientific Center of the World Bank “Vector”), diluted in saline to a concentration of 100,000 microbial cells / reaction. The results were interpreted based on the presence (or absence) of the intersection of the fluorescence curve with the threshold line set at the appropriate level, which corresponds to the presence (or absence) of the threshold cycle Ct in the corresponding column in the results table. The result was considered positive if the fluorescence accumulation curve for the corresponding sample had a characteristic "sigmoid" shape and crossed the threshold line. The Ct value for this sample was not more than 40. The experimental results are shown in table 5.

Table 5. Determination and subtyping of P. multocida in samples of cultures of microorganisms by real-time PCR using panel strains of bacteria of the genus P. multocida (subtypes A, B, D) as panel samples

No.
p / p Subtype
P. multocida Sta
mm The value of Ct / primers and probes for subtypes of P. multocida P. multocida A AT D E F one A 1231 16.28 20.51 neg neg neg neg 2 B 681 13.35 neg 19.68 neg neg neg 3 D T80 15.37 neg neg 18.07 neg neg

Studies (table 4) showed that the calculated and synthesized primers and probes compared to the prototype more specifically detected the bacterium P. Multocida and serotype F 10 times, and serotypes A, B and E 100 times. The use of negative controls (strain 16 M. haemolytica type A1 and strain F-50 E. coli), diluted in physiological saline to a concentration of 100,000 microbial cells / reaction, did not cause the appearance of a fluorescent signal, which indicates the specificity of the used primers and probes.

Claims (1)

A set of oligonucleotide primers and fluorescently-labeled probes for identification and subtyping of DNA of the bacterium Pasteurella multocida serotypes A, B, D, E, F by real-time PCR, characterized in that it contains:
- universal direct (F) and reverse (R) primers and fluorescently-labeled DNA probe (Z) to identify P. multocida bacteria:

- specific direct (F) and reverse (R) primers and fluorescently-labeled DNA probe (Z) to identify serotype A bacteria P. multocida:

- specific direct (F) and reverse (R) primers and a fluorescently-labeled DNA probe (Z) to identify serotype B bacteria P. multocida:

- specific direct (F) and reverse (R) primers and fluorescently-labeled DNA probe (Z) to identify the serotype D of the bacterium P. multocida:

- specific direct (F) and reverse (R) primers and fluorescently-labeled DNA probe (Z) to identify the serotype E of the bacterium P. multocida:

- specific direct (F) and reverse (R) primers and fluorescently-labeled DNA probe (Z) to identify the serotype F of the bacterium P. multocida:

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