EP4320272A1 - Test lamp rapide pour la détection de corynebacterium diphteriae - Google Patents

Test lamp rapide pour la détection de corynebacterium diphteriae

Info

Publication number
EP4320272A1
EP4320272A1 EP22779318.9A EP22779318A EP4320272A1 EP 4320272 A1 EP4320272 A1 EP 4320272A1 EP 22779318 A EP22779318 A EP 22779318A EP 4320272 A1 EP4320272 A1 EP 4320272A1
Authority
EP
European Patent Office
Prior art keywords
primers
seq
gene
diphtheriae
loop
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22779318.9A
Other languages
German (de)
English (en)
Inventor
Shyam Sundar NANDI
Subarna ROY
Upendra LAMBE
Sonali Ankush Sawant
Trupti GOHIL
Manoj MURHEKAR
Jagadish DESHPANDE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Indian Council of Medical Research
Original Assignee
Indian Council of Medical Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Indian Council of Medical Research filed Critical Indian Council of Medical Research
Publication of EP4320272A1 publication Critical patent/EP4320272A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
    • C12R2001/16—Corynebacterium diphtheriae

Definitions

  • the present disclosure relates generally to the field of molecular biology. Particularly, the present disclosure provides novel primers and rapid loop mediated isothermal amplification method for detection of Corynebacterium diphtheriae and differentiating between toxigenic and non- toxigenic strains of C. diphtheriae.
  • Diphtheria is an acute, highly infectious, and potentially lethal disease caused by diphtheria toxin- producing bacterial strains of Corynebacterium diphtheriae.
  • the infection can be transmitted through contact with infected persons and objects that are touched by them.
  • the disease could be presented as respiratory or cutaneous diphtheria, depending on the anatomic site that is affected by the toxigenic Corynebacteria. Rarely other sites can also be affected such as ear, eye, and vulva.
  • Diphtheria toxin absorbed from the mucosal or cutaneous lesions causes toxic damage to the nervous system, myocardium and kidneys. In respiratory diphtheria cases, pseudomembranes in the trachea can cause obstruction in the airways (WHO, 2017).
  • ADS Anti-diphtheritic Serum
  • DAT Dipththeria Anti-Toxin
  • ADS is often not available in rural settings and because of unprofitability production is negligible. Rapid microbiological tests are therefore of high value.
  • clinical diagnosis is not easy and might be confusing with other causes, such as streptococcal sore throat or tonsillitis (ECDC 2020).
  • the current diagnosis methods include isolation of the bacteria on the selective media containing tellurite followed by identification by PCR (CDC 2020) or by classical morphology and biochemical tests. Because of inherent difficulties with culturing, there is high risk of misdiagnosis particularly in countries where the diphtheria is uncommon. In developing countries, facilities for culturing of Corynebacteria in Tellurite media and / or PCR requires specialized / experienced laboratory infrastructure and skills. In all cases, the time taken for completion of a test is more than 24 hours and therefore is of little value to the patient who is recommended to be put on antibiotics and ADS on suspicion anyway. Therefore, the present tests available are of little value in epidemiological purposes in community screening, contact tracing and of course for treatment.
  • Diphtheria is re-emerging in various States. Increasingly more cases are being reported from adults, which, till recently was known to be primarily a childhood disease. Unfortunately, drastic reduction of cases over the years, and perhaps because of the vaccine preventable nature of the disease, an apparent complacency had set in, and the disease went into oblivion in the minds of healthcare professionals and policy makers. Diphtheria is usually diagnosed based on a patient’s clinical presentation.
  • the present invention provides a novel rapid loop mediated isothermal amplification assay for early detection of C. diphtheriae among the patients with typical symptoms such as a thick, gray membrane covering throat and tonsils, sore throat and hoarseness, swollen glands (enlarged lymph nodes), difficulty in breathing or rapid breathing, nasal discharge, fever and chills.
  • the present invention relates to detection of Corynebacterium diphtheriae.
  • the present invention also provides an assay for differentiating between toxigenic and non-toxigenic strains of C. diphtheriae.
  • Fig.l shows interpretation of colorimetric isothermal assay for detection of C. diphtheriae with anticipated results.
  • the invention discloses a loop-mediated isothermal amplification (LAMP) for detection of Corynebacterium diphtheriae and differentiating between toxigenic and non-toxigenic strains of C. diphtheriae.
  • LAMP loop-mediated isothermal amplification
  • the present invention can be applied to the suspected clinical samples that can be used in the field as a rapid detection assay. This invention is easy to perform, rapid and the results can be interpreted visually and does not require any instruments.
  • the present invention involves LAMP technology which uses multiple forward and reverse (backward) primers and one or two loop primers in reaction. Therefore, to design primers for the detection of Corynebacterium diphtheriae, two genes have been targeted by performing bio informatics analysis. It is established that tox gene encoding diphtheria toxin is present only in the pathogenic strains of C. diphtheriae. On the other hand, dtxR gene encoding global regulator is a species-specific gene. The C. diphtheriae species can be detected by using species specific
  • the gaps shown in the sequences 3 and 4 in the above table is the representative of the F2/B2 and Flc/Blc which is the part of FIP/BIP primers.
  • the gene dtxR For each gene, two sets of primers have been designed.
  • LAMP requires a modified Bst 2.0 DNA polymerase having strand displacement activity. LAMP reactions can therefore be carried out at a single incubation temperature (isothermal).
  • primers for the detection of Corynebacterium diphtheriae in a sample have been provided, wherein primers having Sequence IDs 1-10 are designed against tox gene of Corynebacterium diphtheriae and primers having SEQ IDs 11-20 are designed against dtxR gene of Corynebacterium diphtheriae.
  • primers having SEQ IDs of 1-5 are designed against tox gene.
  • primers having SEQ IDs of 6-10 are designed against tox gene.
  • primers having SEQ IDs of 11-15 are designed against dxtR gene.
  • primers having SEQ IDs of 16-20 are designed against dtxR gene.
  • a rapid loop mediated isothermal amplification method for detection of Corynebacterium diphtheriae and differentiating between toxigenic and non- toxigenic strains of C. diphtheriae comprises the steps of: i) isolating DNA from a sample; ii) amplifying said DNA through LAMP amplification method involving a primer system, wherein said system comprising: outer primer pairs: forward outer primers and backward outer primers (F3/B3); inner primer pairs : forward inner primers and backward inner primers (FIP/BIP) and loop primer pairs: loop forward primer and loop backward primers (LF/LB); iii) detecting the amplified DNA by detecting the dtxR and Tox gene by change of colour.
  • forward outer primers are selected from SEQ ID No. 1, 6, 11 and 16.
  • backward outer primers are selected from SEQ ID No. 2, 7, 12 and 17.
  • forward inner primers are selected from SEQ ID No. 3, 8, 13 and 18.
  • backward inner primers are selected from SEQ ID No. 4, 9, 14 and 19.
  • loop primer pairs are selected from SEQ ID No. 5, 10, 15 and 20.
  • said method is conducted at 65°C for 30 minutes.
  • LAMP technology uses multiple forward and reverse (backward) primers and one or two loop primers in reaction.
  • LAMP requires a modified Bst 2.0 DNA polymerase having strand displacement activity which is responsible for isothermal amplification of DNA.
  • the template DNA strand along with Bst 2.0 DNA polymerase and primers is incubated at a constant temperature of 60°C-65°C. The results are interpreted in the form of color change form pink to yellow.
  • LAMP primers set contains a) F3/ B3 (Forward/ Backward outermost primers) which are similar to PCR primers, b) FIP/ BIP (Forward/ Backward inner primers), FIP and BIP are specialized primers with complementary and non-complementary nucleotide segments and c) LF or LB (forward/ backward loop primers) or both.
  • FIP and BIP are specialized primers consisting of two parts, F2/Flc and B2/Blc, respectively.
  • F2 and B2 bind with the template strand to initiate amplification process while Flc and Blc sequences serve as overhangs which help loop formation as LAMP reaction continues.
  • the short distance between the F2 and Flc (and B2/ Blc) helps formation of a loop structure within the amplicon.
  • the loop primers increase the number of initiation points for DNA synthesis by binding complementarily to the single stranded loops and increase the pace of amplification.
  • the first gene dtxR is a species specific gene for C. diphtheriae (Holmes 2000). By using this gene C. diphtheriae species can be identified from the clinical samples. On the other hand, to detect the toxin producing or pathogenic C. diphtheriae strains tox gene is targeted (Zasada et al., 2020).
  • the four key factors in the LAMP primer design are the melting temperature (Tm), stability at the 3’ and 5’ end of each primer (Delta G), GC content and ability to form secondary structures.
  • Tm is calculated by using the Nearest-Neighbor method. This method is presently considered to be the method that predicts the Tm value closest to the actual value.
  • the Tm for each region is designed to be about 65°C (64 - 66°C) for Flc and Blc, about 60°C (59 - 61 °C) for F2, B2, F3, and B3, and about 65°C (64 - 66°C) for the loop primers.
  • the 3’ end of the primers acts as the initiating point of the DNA polymerization and therefore, must be very stable and complementary with the target sequence. The following criteria are taken into consideration while selecting the primer sets.
  • the 3’ ends of F2/B2, F3/B3 are designed so that the free energy is -4 kcal/ mol or less.
  • LF/LB and the 5’ end of Flc/Blc are designed so that the free energy is -4 kcal/ mol or less.
  • Primers are designed so that their GC content is in between about 40% to 65%. But, primers with GC content between 50% and 60% are selected as they are considered to give relatively better results.
  • primers Another important property of primers is the ability to form secondary structures or primer dimers.
  • the primers are designed so that they do not form secondary structures. To avoid this condition, it is important to make sure that the 3’ end of the primer is non complementary.
  • a primer mixture is made prior to the actual LAMP reaction.
  • the primer mixture consists of 1.6 mM of FIP and BIP, 0.2 mM of F3 and B3, and 0.4 mM of LF and LB (Tablel).
  • the assay was performed in a 20 m ⁇ reaction mixture containing 2 m ⁇ of 10 X primer mixture of 16 mM of both forward inner primer (FIP) and backward inner primer (BIP); 2 mM of both F3 and B3 primers and 4 mM of both loop forward (LF) and loop backward (LB). 10 m ⁇ of WarmStart
  • LAMP consists of the following components mentioned in the table 2.
  • the tubes are removed from the incubator and observed with the naked eye.
  • the positive reaction is indicated by yellow colored reaction mix.
  • the color of negative reaction remains pink ( Figure 1). If the color change is not significant, for example if the color of reaction is orange, incubate the reaction at 65° C for more 10 minutes and re-examined for the complete color change.
  • the color of reaction gets intensified by cooling the reaction to cool down at room temperature.
  • Novelty of the innovation All the primers for LAMP assay are new and novel and designed.
  • This disclosure relates to a LAMP based assay for the detection of C. diphtheriae. Detection of C. diphtheriae using isothermal amplification method which does not require a thermal cycler as in case of real-time PCR.
  • the assay can be performed using a single temperature heating device (65 ⁇ 1° C). The time required for the assay is only 35 to 40 minutes. The results can be interpreted visually; no other sophisticated instruments are required for interpretation of results. Highly skilled or technical person is not required to perform this assay. It is cost effective and less time-consuming technology. This assay could enable point-of-care testing outside of the diagnostic laboratory.
  • ECDC European Centre for Disease Prevention and Control

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une nouvelle amorce et un procédé d'amplification isotherme à médiation par boucle rapide pour la détection de Corynebacterium diphtheriae. En outre, la présente invention concerne également un test pour la différenciation entre des souches toxigènes et non toxigènes de C. diphtheriae.
EP22779318.9A 2021-03-31 2022-03-22 Test lamp rapide pour la détection de corynebacterium diphteriae Pending EP4320272A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN202111015391 2021-03-31
PCT/IN2022/050276 WO2022208531A1 (fr) 2021-03-31 2022-03-22 Test lamp rapide pour la détection de corynebacterium diphteriae

Publications (1)

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EP4320272A1 true EP4320272A1 (fr) 2024-02-14

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EP22779318.9A Pending EP4320272A1 (fr) 2021-03-31 2022-03-22 Test lamp rapide pour la détection de corynebacterium diphteriae

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4876223B2 (ja) * 2006-02-28 2012-02-15 財団法人ヒューマンサイエンス振興財団 Ｌａｍｐ法を用いたジフテリア毒素遺伝子検出方法およびこの方法に用いるプライマーセット
US10724091B1 (en) * 2015-02-10 2020-07-28 National Technology & Engineering Solutions Of Sandia, Llc Endpoint detection of amplified nucleic acids
EP3805407A4 (fr) * 2018-05-25 2021-08-11 Mitsubishi Chemical Corporation Procédé de quantification d'acides nucléiques cibles

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EP4320272A1 (fr) Test lamp rapide pour la détection de corynebacterium diphteriae

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