Development and validation of loop-mediated isothermal amplification (LAMP) for detection of leptospir genetic material

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National scientific center «Institute of experimental and clinical veterinary medicine»

State scientific and control institute of biotechnology and strains of microorganisms

Development and validation of loop-mediated isothermal amplification (LAMP) for detection of leptospir genetic material

Korobka A.I.

Arefiev V.L.

Kharkiv

Kyiv

Abstract

Primer systems were designed and tested and the method of loop-mediated isothermal amplification was validated for rapid diagnosis of leptospirosis. In general, five sets of primers were calculated and analyzed in situ and in vitro, which were ordered for synthesis and chosen for the future as the most promising. Indicators of analytical sensitivity and detection limits of the method were established, the specificity of the method in its tests with homologous and close to taxonomic characteristics heterologous samples of genetic material was proved and completed reproducibility of the method was established under the conditions of its staging on various laboratory equipment and with reagents from different manufacturers. The developed technique of loop-mediated amplification for the diagnosis of leptospirosis was tested on field samples and its diagnostic effectiveness was established.

Key words: DNA, leptospirosis, loop-mediated isothermal amplification, primer system

Анотація

Розроблення та валідація петлевої ізотермальної ампліфікації (LAMP) для детекції генетичного матеріалу лептоспір

Коробка А.І^., Ареф'єв В.Л.

Національний науковий центр «Інститут експериментальної і клінічної ветеринарної медицини», Харків, Україна;

Державний науково-контрольний інститут біотехнології і штамів мікроорганізмів, Київ, Україна;

Сконструйовано та випробувано праймерні системи та проведено валідацію методики ізотермальної петлевої ампліфікації для експрес діагностики лептоспірозу. Загалом розраховано та проаналізовано insituта invitroп'яти сетів праймерів, що були замовлено для синтезу та обрано для подальших найбільш перспективні. Встановлено показники аналітичної чутливості, межі детекції методики, доведено специфічність методики у випробуваннях її з гомологічними та близькими за таксономічними характеристиками гетерологічними зразками генетичного матеріалу, встановлена повна відтворюваність методики за умов її постановки на різному лабораторному обладнанні та з реагентами різних виробників. Розроблена методика петелевої ампліфікації для діагностики лептоспірозу випробувана на польових зразках та встановлена її діагностична ефективність.

Ключові слова: ДНК, лептоспіроз, петлева ізотермальна ампліфікація, система праймерів

Main part

Leptospirosis is considered one of the most common natural-focal zoonotic infections of wild, domestic animals and humans (Bharti et al., 2003). Clinical leptospirosis is common in dogs that can excrete leptospira with the urine in the absence of signs of disease.

Laboratory diagnostics of leptospirosis are based on outdated and complicated diagnostic methods (MAT, microscopy in the dark field). In addition to serological methods of research, methods of molecular biology have recently been used - polymerase chain reaction in the classical version and in real-time. These methods have great practicality in routine work, because bacteriological studies in leptospirosis, particularly the isolation of pure culture from field material, are problematic and timeconsuming.

The leptospirosis control system requires today additional express methods of laboratory diagnostics. One such area is the establishment of test systems based on loop-mediated isothermal amplification (LAMP) (Tsugunori et al., 2000). This method allows to identify the DNA of leptospirosis pathogens more effectively than PCR diagnostics, and does not require special expensive equipment. First of all, LAMP continues at a constant temperature mode, so only any simple device that can maintain a constant temperature is needed to implement it - a thermostat, a water bath, etc. The results can be obtained with the field condition using portable ultraviolet radiation detectors. The main advantage of this method is higher sensitivity and specificity compared to traditional methods of molecular genetic studies.

Today, in Ukraine there is an urgent need for a comprehensive study of the leptospirosis problem, which should consist of seromonitoring, the establishment of serological groups of pathogens that circulate in different regions, and it is also necessary to present new more effective systems for rapid diagnosis of leptospirosis to improve the effectiveness of surveillance for these diseases.

The main purpose is to develop and validate loop-mediated isothermal amplification for the detection of leptospira DNA in clinical material samples.

Material and methods. The PUBMED site was used to generate databases about sequences. Nucleotide sequences were stored in FASTA format. PrimerExplorer V5 (http://primerexplorer.jp/e) was used to calculate the appropriate primers. Validation of the isothermal amplification method was carried out according to the standard scheme, which consists of determining the following indicators:

Analytical sensitivity - determine the minimum concentration of the DNA matrix in the sample that the test system can identify.

Specificity - determine the ability to exactly and selectively detect a certain substance in the presence of components that may occur in the DNA matrix (impurities, similar chemical compounds).

Limit of detection - using a DNA sample of known concentration, a value was determined close to analytical sensitivity that the test system can identify and 10 samples of each dilution are tested. The experiment was repeated twice on different days to determine the limit of detection - the minimum concentration of the pathogen, which gives at least 95% of positive results (at least 29 out of 30).

Evaluation of reproducibility - reproduced the method under different conditions, on different equipment and using reagents from different manufacturers. To determine the diagnostic effectiveness, positive and negative field DNA samples were isolated from the dog's urine that were selected during the expedition trips. A total of 1,025 DNA samples were tested.

Results. According to the literature data for the construction of oligonucleotide sequences for conducting PCR, the most promising leptospira genes are flaB, ompLI, lipL25, ligA, ligB, lipL32, lipL41 та16S ribosomal RNA (rrs) (Gentilini et al, 2017; Hsu et al., 20l7; Chen et al., 2015; Suwancharoen et al, 2016). These genes are highly conserved within the species Leptospira interrogans, allowing them to be used to design specific primers. To do this, the sequences of these genes were loaded from the GenBank database for the next multiple sequence alignment using the BioEdit program to determine the relevant regions. Thus, the base of nucleotide sequences of the genes was formed such as LipL32 (n = 58), LipL41 (n = 54), rrs (n = 69), ompL1 (n = 48), flaB (n = 47). When processing other genes, it turned out that their length does not comply with the requirements that are applied when developing primers for conducting LAMP.

At the next stage, the selected genes were investigated to search for conservative regions and selection within appropriate sites for calculating primers. Thus, within the rrs gene, 4 regions were selected, and LipL32, LipL41, ompL1, flaB - three. At the same time, it turned out that reverse primers were impossible to select because of the short distances between such regions in the flaB and ompL genes.

To calculate primers within LipL32/LipL41 genes and their sequence was analyzed using the PrimerExplorer V5 online resource (fig. 1).

A panel of positive control DNA samples was manufactured for the investigation. To do this, 14 - day leptospira cultures of the diagnostic line (13 serogroups) were cultivated. To increase the purity of the samples, and to get rid of the nutrient medium components, they were washed three times in saline solution by centrifugation and dilution of the precipitate. At each washing step, the presence of live leptospira was monitored by dark-field microscopy. Further, DNA was isolated from the obtained pure leptospira samples by sorbent sorption using the commercial kit of the QIAGEN campaign. The concentration of the obtained DNA was measured on a NanoDrop spectrophotometer, and the obtained data were fixed for further calculations of validation parameters (Table 1).

Figure 1. Selection of primer sequences to develop a method for detecting leptospira genetic material based on LAMP

According to the results of bioinformatic calculations, the following primary systems were selected for synthesis, the list of which consisted of five sets:

Set 1:

¦ One Health Journal. 2024. Vol. 2. №2

TGTTTGGATTCCTGCCGTAA

GCAGCTTTGGCGATTTGG

TAAGTCTCCGTCGCCTGGCTC-TCGCTGAAATGGGAGTTCG AGCGGCTACCCCAGAAGAAAAA-AGGCATAATCGCCGACATTC TTCGCCTGTTGGGGAAATCATA GGTTT GATACTTGGATCCGT GTAGA

Set 2:

Lip32_2_F3 CCAGGGACAAACGAAACCG Lip32_2_B3 GCTTACTAAGTCTCCGTCGC

Lip32_2_FIP TAAACCGTCCGGCGCTTGTCACTTCCCTACGGATCTGTGA Lip32_2_BIP TGGATTCCTGCCGTAATCGCTGCTCACCGATTTCGCCTGTT Lip32_2_LF TGGCTTTACGTATCCGTAATAGTTG Lip32_2_LB AATGGGAGTTCGTATGATTTCCC

Set 3:

Lip32_3_F3 TGTTTGGATTCCTGCCGTAA Lip32_3_B3TTTTGCTTTCGCAGCTTTGG

Lip32_3_FIP CGCTTACTAAGTCTCCGTCGCCCGCTGAAATGGGAGTTCGT Lip32_3_BIP AGCGGCTACCCCAGAAGAAAAAAGGCATAATCGCCGACATTC Lip32_3_LF TTTCGCCTGTTGGGGAAATCAT Lip32_3_LBAATGCCACATTGGTTT GATACTTGG

Set 4:

Lip32_4_F3 AAGCATACTATCTCTATGTTTGG Lip32_4_B3TTGGTCAGGCATAATCGC

Lip32_4_FIP GCTTACTAAGTCTCCGTCGCCCGTAATCGCTGAAATGGGA Lip32_4_BIP GACGCTTTCAAAGCGGCTACTTCTTTCTACACGGATCCAAG Lip32_4_LF GCCTGTTGGGGAAATCATACG Lip32_4_LBCCCAGAAGAAAAAT CAATGCCACA

Set 5:

Lip32-5-F3 TCTATGTTTGGATTCCTGCC Lip 32-5-B3 ATCGT CACCAT CAT CAT CAT C

Lip 32-5-FIP CGCTTACTAAGTCTCCGTCGCGTAATCGCTGAAATGGGAGT Lip 32-5-BIP GCGGCTACCCCAGAAGAAAAGCATAATCGCCGACATTCT Lip 32-5-LF CTCACCGATTTCGCCTGT Lip 32-5-LB TGCCACATTGGTTTGATACTTG

Table 1. Primary concentration leptospiral DNA after isolation