EP4320272A1

EP4320272A1 - Rapid lamp assay for detection of corynebacterium diphtheriae

Info

Publication number: EP4320272A1
Application number: EP22779318.9A
Authority: EP
Inventors: Shyam Sundar NANDI; Subarna ROY; Upendra LAMBE; Sonali Ankush Sawant; Trupti GOHIL; Manoj MURHEKAR; Jagadish DESHPANDE
Original assignee: Indian Council of Medical Research
Current assignee: Indian Council of Medical Research
Priority date: 2021-03-31
Filing date: 2022-03-22
Publication date: 2024-02-14
Also published as: WO2022208531A1

Abstract

The present invention relates to a novel primers and rapid loop mediated isothermal amplification method for detection of Corynebacterium diphtheriae. Further, the present invention also provides an assay for differentiating between toxigenic and non-toxigenic strains of C. diphtheriae.

Description

Rapid LAMP assay for detection of Corynebacterium diphtheriae

TECHNICAL FIELD:

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.

BACKGROUND ART:

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).

The infections caused by toxigenic Corynebacteria seemed to be well controlled in developed countries since the introduction of vaccination against diphtheria in the 1940s. However, infections recorded during the last several years point at C. diphtheriae and C. ulcerans as re-emerging human pathogens. Domestic pets and other animals have been described as reservoirs and sources of diphtheria infection (Zasada et al., 2020). A number of reports of either re-emergence or persistence of diphtheria from several Indian states, including Karnataka, Andhra Pradesh, Delhi, Maharashtra, Chandigarh, Gujarat, Assam, West Bengal, Madhya Pradesh, Uttar Pradesh, Rajasthan and Haryana have been reported (Parande et., al. 2014; Parande et ah, 2017; Murhekar 2017). As per the CBHI data, during 2005-2014, India reported 47524 cases (average: 4752 per year) with 1314 deaths (case fatality ratio: 2.8%) (CBHI 2019).

Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. This is because administration of antibiotics is not enough to treat the patient, as the very potent Diphtheria toxin must be neutralized with Anti-diphtheritic Serum (ADS) also called Dipththeria Anti-Toxin (DAT). ADS is often not available in rural settings and because of unprofitability production is negligible. Rapid microbiological tests are therefore of high value. Furthermore, 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. Therefore, development of a point-of-care test for diphtheria is of prime importance especially in developing countries like India where access to medical laboratories is extremely limited as are investigations of an infection source (ECDC 2020). Also because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts (Zasada et ah, 2020). The importance of development of a point of care diagnostic test for diphtheria assumes a necessity, particularly for addressing neglected diseases that affect the poor and deprived.

After a steady decline in global cases of diphtheria following widespread immunization since the 1980s annual incidence of diphtheria reached a plateau and has not shown any further sign of reduction in the last decade and half since 2006. India alone has accounted for almost 80% of these cases. 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. Since laboratory demonstration of presence of Corynebacterium diphtheriae in the throat swab samples require culture and isolation or PCR based tests that require time. Since culture and isolation of C. diphtheriae is difficult and takes 2- 3 days’ time, PCR is often used for demonstrating presence of tox gene capable of producing the Diphtheria toxin in the clinical specimens infected with toxigenic C. diphtheriae. PCR is also costly and takes more than a day since it is normally carried out after culturing the swabs on suitable media. It is also expensive, require expertise and can be performed only in high-end laboratories. Therefore, the available diagnostic tests cannot be applied for contact tracing and diagnosis in field conditions.

In order to address problem of the art, 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.

SUMMARY:

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.

BRIEF DESCRIPTION OF THE DRAWINGS:

These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings wherein:

Fig.l shows interpretation of colorimetric isothermal assay for detection of C. diphtheriae with anticipated results.

DETAILED DESCRIPTION:

For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the figures and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.

The terminology and structure employed herein is for describing, teaching and illuminating some embodiments and their specific features and elements and does not limit, restrict or reduce the spirit and scope of the invention.

Definitions

For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person skilled in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below. The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”. Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference. The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purposes of exemplification only. Functionally, equivalent products and methods are clearly within the scope of the disclosure, as described herein.

Aspects and embodiments of the present invention will be described below in detail with reference to the accompanying drawings.

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. The laboratory demonstration of presence of C. diphtheriae in the throat swab samples require culture and isolation or PCR based tests. Since culture and isolation of C. diphtheriae is difficult and takes 2- 3 days’ time while PCR is also costly and takes more than a day since it is normally carried out after culturing the swabs on suitable media. Additionally, it requires expertise and can be performed only in high-end laboratories.

Therefore, the currently available diagnostic tests cannot be applied for surveillance and detection at the point of care. 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. In addition to this, 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). Accordingly, in an aspect of the present invention 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.

In an embodiment, primers having SEQ IDs of 1-5 are designed against tox gene.

In another embodiment, primers having SEQ IDs of 6-10 are designed against tox gene.

In yet another embodiment, primers having SEQ IDs of 11-15 are designed against dxtR gene.

In a second embodiment, primers having SEQ IDs of 16-20 are designed against dtxR gene.

In another aspect of the present invention, a rapid loop mediated isothermal amplification method for detection of Corynebacterium diphtheriae and differentiating between toxigenic and non- toxigenic strains of C. diphtheriae has been provided, wherein said method 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.

In an embodiment, forward outer primers are selected from SEQ ID No. 1, 6, 11 and 16.

In another embodiment, backward outer primers are selected from SEQ ID No. 2, 7, 12 and 17.

In yet another embodiment, forward inner primers are selected from SEQ ID No. 3, 8, 13 and 18.

In one more embodiment, wherein backward inner primers are selected from SEQ ID No. 4, 9, 14 and 19. In an embodiment, loop primer pairs are selected from SEQ ID No. 5, 10, 15 and 20.

In another embodiment, said method is conducted at 65°C for 30 minutes.

EXAMPLES:

The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.

Example 1:

LAMP:

LAMP technology uses multiple forward and reverse (backward) primers and one or two loop primers in reaction. In addition to this, 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.

Primers: 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.

For this assay two genes of C. diphtheriae have been targeted. 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).

Characteristic properties of primers:

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:

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 stability of the 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.

GC content:

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.

Secondary structures:

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.

Example 2:

Methodology:

The DNA isolated from clinical cases of Diphtheria in Raichur district collected by MRHRU were used for the standardization. A commercially available kit is used for LAMP; WarmStart® Colorimetric LAMP 2X Master Mix with UDG (DNA & RNA) (Catalogue number M1800L).

Primer mixture:

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).

Tablel: Concentration each primer to be incorporated in the primer mixture. LAMP reaction mixture:

The assay was performed in a 20 mΐ reaction mixture containing 2 mΐ 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 mΐ of WarmStart

Colorimetric LAMP 2X Master Mix with UDG (M1804L), 0.8 mΐ of enhancer, 3.2 mΐ of nuclease free water and 4 mΐ of DNA template. The reaction mixture was mixed thoroughly by vortex followed by a quick spin. Later the reaction was set at 65°C for 30 minutes on a water bath. The results were observed with naked eye by looking at the color change. The reaction mixture for

LAMP consists of the following components mentioned in the table 2.

Table 2: The composition of LAMP reaction

Interpretation of results:

The tubes are removed from the incubator and observed with the naked eye. The positive reaction is indicated by yellow colored reaction mix. On the other hand, 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.

It will thus be seen that the objects set forth above, among those made apparent from the preceding description, are efficiently attained, and since certain changes may be made in the constructions set forth without departing from the spirit and scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. The invention has been described with reference to preferred and alternate embodiments. Modifications and alterations will become apparent to those skilled in the art upon reading and understanding the detailed discussion of the invention provided herein. This invention is intended to include all such modifications and alterations insofar as they come within the scope of the present invention. These and other modifications of the preferred embodiments as well as other embodiments of the invention will be obvious from the disclosure herein, whereby the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

Finally, to the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.

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.

References:

• World Health Organization. Diphtheria vaccine: WHO position paper-August 2017-

Vaccin antidiphterique: Note de synthese de TOMS-aout 2017. Weekly Epidemiological Record= Releve epidemiologique hebdomadaire. 4;92(31):417-36.

• European Centre for Disease Prevention and Control (ECDC). Gap analysis on securing diphtheria diagnostic capacity and diphtheria antitoxin availability in the EU/EEA. Stockholm: ECDC; 2017.

• Parande MV., Parand AM., Lakkannavar SL., Kholkute SD, Roy S. Diphtheria outbreak in rural north Karnataka, India. JMM Case reports 2014; 1 (3), e003558

• Parande MV., Roy S., Mantur BG., Parande AM., Shinde RS. Resurgence of diphtheria in rural areas of North Karnataka, India. Indian Journal of Medical Microbiology. 2017. 35 (2), 247

• Murhekar M. Epidemiology of diphtheria in India, 1996-2016: implications for prevention and control. The American journal of tropical medicine and hygiene. 2017 Aug 2;97(2):313-8.

• Zasada AA, Wiatrzyk A, Czajka U, Brodzik K, Formmska K, Mosiej E, Prygiel M, Krysztopa-Grzybowska K, Wdowiak K. Application of loop-mediated isothermal amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for Corynebacterium diphtheriae. BMC Infectious Diseases. 2020 Dec;20:l-9.

• Central Bureau of Health Intelligence (CBHI), Govt of India. National Health Profile 2005. Onwards Available at: http://www.cbhidghs.nic.in/indexl. asp?linkid=267. Accessed April 15, 2017

• Centers for disease control and prevention (CDC) (2020). Diphtheria: Diagnosis and treatment. Available at: https://www.cdc.gov/diphtheria/about/diagnosistreatment. html. Accessed on 20th October 2020

• Holmes RK. Biology and molecular epidemiology of diphtheria toxin and the tox gene. Journal of Infectious Diseases. 2000 Feb l;181(Supplement_l):S156-67.

• Atkinson W. (2006) Epidemiology and prevention of vaccine -preventable diseases. Department of Health & Human Services, Centers for Disease Control and Prevention; 215-230

Claims

We claim:

1. Primers for the detection of Corynebacterium diphtheriae in a sample, 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.

2. Primers as claimed in claim 1 , wherein primers having SEQ IDs of 1 -5 are designed against tox gene.

3. Primers as claimed in claim 1, wherein primers having SEQ IDs of 6-10 are designed against tox gene.

4. Primers as claimed in claim 1, wherein primers having SEQ IDs of 11-15 are designed against dtxR gene.

5. Primers as claimed in claim 1, wherein primers having SEQ IDs of 16-20 are designed against dtxR gene.

6. A rapid loop mediated isothermal amplification method for detection of Corynebacterium diphtheriae and differentiating between toxigenic and non-toxigenic strains of C. diphtheriae, wherein said method comprises the steps of: iv) isolating DNA from a sample; v) 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); vi) detecting the amplified DNA by detection of tox and dtxR gene by change of colour.

7. The method as claimed in claim 6, wherein forward outer primers are selected from SEQ ID No. 1, 6, 11 and 16.

8. The method as claimed in claim 6, wherein backward outer primers are selected from SEQ ID No. 2, 7, 12 and 17.

9. The method as claimed in claim 6, wherein forward inner primers are selected from SEQ ID No. 3, 8, 13 and 18.

10. The method as claimed in claim 6, wherein backward inner primers are selected from SEQ ID No. 4, 9, 14 and 19.

11. The method as claimed in claim 6, wherein loop primer pairs are selected from SEQ ID No. 5, 10, 15 and 20.

12. The method as claimed in claim 6, wherein said method is conducted at 65°C for 30 minutes.