ES2477441B1 - Use of restriction enzymes as molecular markers for the diagnosis of human Trichuriasis and other Trichuriasis of animal origin - Google Patents

Abstract

Use of restriction enzymes as molecular markers for the diagnosis of human Trichuriasis and other Trichuriasis of animal origin consisting of a specific procedure based on molecular markers to differentiate which species of Trichuris is infecting man, or which species of Trichuris is present in any sample of feces, waters, or others; to be applied to the diagnosis, epidemiology, control and prophylaxis, and treatment of human and animal trichuriasis.

Description

Use of restriction enzymes as molecular markers for the diagnosis of human Trichuriasis and other Trichuriasis of animal origin. 5

OBJECT OF THE INVENTION

The object of the invention, as expressed in the statement of this specification, is the use of a Trichuris 10 specific gender differentiation procedure based on specific molecular markers to be applied to diagnosis, epidemiology, control and prophylaxis, and treatment of human and animal trichuriasis.

The scientific area to which the invention corresponds would be that of ~ molecular parasitology "and the Activity Sector would be a priority in the pharmaceutical sector and, in general, in the health sector. That is, the same would be applicable in the field of health for the diagnosis of trichuriasis, in the field of epidemiology to carry out studies of prevalence, incidence, zoonosis, as well as in the detection of Trichuris eggs in the analysis of wastewater, in order to carry better and Reliable control measures, as well as in the field of the pharmaceutical industry for the development of new

20 therapies (vaccines) for trichuriasis and in Crohn's disease therapies.

BACKGROUND IN THE STATE OF THE TECHNIQUE

Gastrointestinal helminth parasites are considered as infections

25 most common parasites. In fact, more than a quarter of the world's population is infected, being, of chronic human infections of any kind, the most prevalent. Most of these infections are attributed to four species of nematodes, each infecting hundreds of millions of people. These species, in order of abundance, are: Ascaris / umbricoides, Trichuris trichiura, Necator

30 americanus and Ancy / ostoma duodena / e. Although the extraordinary abundance of these nematodes has been extensively investigated for many years, the global recognition of the sanitary importance of these infections was not established until 1960, when the World Health Organization (WHO) reactivated a research and control program for these gastrointestinal helminth infections.

The species of the genus T richuris are parasitic nematodes of the cecum of different hosts. They have a direct biological cycle; Once the worm settles in the epithelium of the intestinal lumen, it begins to mature and grows, moving through four larval states to adulthood. In this state, the worm penetrates the epithelium through its anterior part, leaving the posterior end free in the lumen. The adult worm seems to feed on enterocltic material and blood, being responsible for a disease in man known as trichuriasis. Trichuriasis is a parasitism that affects a billion people in the world through temperate and tropical areas. Its etiologic agent, Trichuris trichiura, is a parasite of the mucosa of the human colon in which it produces inflammation. Trichuriasis is probably the second most common human parasitic infection in tropical regions. However, it would be a mistake to consider T. trichiura as an exclusively tropical parasite, and thus, although the infection is, today, more prevalent in hot and humid tropical regions, it also occurs in areas other than the tropics.

Together with these epidemiological data, we have the fact that zoonotic infection in Trichuris seems possible, and this is evidenced by the citations of human infection with the dog's lizard (T. vu / pis) (Burrows and Lillis, 1960; Harper and col., 1964; Kenney and Yennakov, 1980; Mirdha and coL, 1998; Dunn et al., 2002) or mixed infections with both species (Vazquez and coL, 1997). However, zoonotic infection with pig tricide (T. suis) seems much more likely, since accidental infections and in human volunteers who have been infected with T. su; s indicate that the latter species is capable of producing eggs completely viable for some months (Beer, 1976). Asr, in many locations where pigs live near humans, zoonotic infection is very likely to occur. The specific differentiation of Trichuris trichiura, T. suis and related species eggs is based on the size of the egg. However, Yoshikawa et al. (1989) mentioned the presence of Trichuris trichiura eggs with two different sizes obtained from the uterus of adult worms. On the other hand, it should be noted that some anthelmintics have an effect on the reproductive system of females of T. trichiura, thus favoring the production of

abnormal eggs, which could lead to a bad diagnosis of the infection, since

they can be confused with the eggs of other parasites or artifacts (Ferrer-Rodríguez and

Kozek, 2007). In this way, so far, there is no characteristic or technique

that differentiates the eggs of the different species of Trichuris, except for the fact

S

find or isolate from a specific host.

On the other hand, the species of the genus Trichuris are among the most commonly

detected in Spanish wastewater. The eggs of the parasite causing the

Human trichuriasis, Trichuris tn'chiura are morphologically almost identical to those of

10

Trichuris suis and are considered by the World Health Organization (WHO) as

one of the three preferred bioindicators to assess the quality of a regenerated water.

Hence the importance of a good and reliable specific determination.

Currently, species of the genus Trichuris have been cited as helminths with some

fifteen

suitable characteristics to be used in the therapy of autoimmune diseases,

such as Crohn's disease, multiple sclerosis and type I diabetes (Elliott and coL,

2007). These diseases result from tissue destruction due to a process

inflammatory, and are prevalent in industrialized countries, highly developed, being

Few in underdeveloped countries. This fact seems to be in relation to the

twenty

human exposure to helminths in underdeveloped countries and to scarce or total

absence of these in the more developed countries. helminths decrease

immune response in humans that are naturally parasitized and reduces the

Inflammation in experimental colitis. Thus, exposure to helminths can help

prevent or even alleviate Crohn's disease. In this regard, they have been carried out

25

some essays by various authors (Sumners et al., 2005, Kradin et al., 2006)

assessing the efficacy and safety of the treatment of Crohn's disease with the

intestinal helminth Trichuris suis. the results obtained indicate that this new

Therapy can offer a safe, effective and unique alternative in Crohn's disease.

These same results also support the premise that natural exposure to

30

helminths such as Trichuris suis provides protection to diseases

immunological like Crohn's disease.

Trichuris species have many characteristics that make them good candidates

For clinical use. Specifically, T. suis has additional features that make it a

attractive candidate: it can be obtained in large quantities with absolute purity of pigs raised in a specific pathogen-free environment and does not cause human disease, and therefore decreases the risk of its use.

There are currently several examination techniques to eliminate the presence of parasite eggs in faecal samples, including direct fecal examination, the technique of the concentration of formol-ether (Ridley and Hawgood, 1956; Allen and Ridley, 1970), the technique of Kato-Katz (Katz et al., 1972; Peters et al., 1980), the McMaster technique (Who, 2008), and the recently described technique of FLOTAC® (Cringoli, 2006). Among them, the Kato-Katz technique (Peters et al., 1980) is the most commonly used (Knopp, 2008; Goodman et al., 2007; Utzinger and coL, 2008), due to the ease of use in the field and relatively low cost. The Kato-Katz technique is recommended by the World Health Organization (WHO) for surveillance and epidemiological surveys of geohelminthiasis (Who, 1994; Montresor et al., 1998). Several authors have cited that the presence of eggs in fecal daily samples varies (Hall, 1982; Anderson and Schad, 1985; Marti and Koella, 1993; Booth et al "2003) and, therefore, the sensitivity of the diagnostic technique used to determine trichuriasis is limited to the day of sampling for the diagnosis of infection.Asl, several authors (Utzinger et al., 2008, Booth et al., 2003) recommended the analysis of faecal samples for consecutive days to improve the sensitivity of the test, but the higher the sensitivity, the lower the specificity of this technique.

To date, the identification and classification of many parasitic species has been based almost exclusively on morphological criteria and, less frequently, on the specificity of the parasite to its host. However, taking into account the degree of morphological variability of the parasites, and the influence that the host or other factors can exert on this variation, the need to evaluate other criteria in taxonomic and diagnostic studies has been reached. Therefore, better characterization methods are required for a more precise definition of parasites of man and animals, as well! as possible reservoir hosts. Moreover, a better understanding of the genetic diversity of parasitic organisms is required since many helminths, which are morphologically identical, appear to have different factors such as infectivity, pathogenicity, immunogenicity and drug sensitivity.

Molecular techniques have been applied for the differentiation of the species of the genus Trichuris (Oliveros and coL, 2000; CutiUas and coL, 2002, 2004 and 2007) based on the amplification and sequencing of the ribosomal segment comprising the transcribed internal spaces (lTS ) 1 and 2 and the 5.88 gene. The result of the study carried out by Oliveros et al. (2000) based on IT82 was the identification of synonyms between Trichuris avis and Trichuris g / abulosa and the observation of an allo percentage of variability between the species T richuris avis, T. suis and T. leporis confirming the diagnostic value of these techniques . The study carried out in 2002 based on the ribosomal fragment IT81-5,8810 IT82 made it possible to distinguish between two different species of trichurids (Trichuris muris and T. arvicolae) in 3 different rodent hosts. Then, in 2004, different populations of T. ovis and T. globulosa, T. leparis and

T. skrjabini. The result was the identification of synonyms between T. avis and T. globulosa.

15 The granting of a Project (CGL2004-00630 / BOS) in 2004 allowed us to carry out a morphological, biometric and molecular study of Trichuris suis isolated from different hosts (pig and wild boar), from Trichuris vulpis isolated from the dog and Trichuris isolated trichura of the monkey and of the man, in which molecular techniques of amplification of AON were tested by means of PCR to the study of the epidemiology and control of the

20 trichuriasis, based on the sequences obtained from the ITS1-5,8S-ITS2 fragment of the ribosomal AON.

The results published in our work (Parasitology Research, 2007) drew attention to groups that worked in the therapy of [Crohn's disease, because it is precisely 25, a therapy based on the administration of Trichuris suis eggs. Thus, Dr. Bernhard Tewes of Falk Pharma GMBH, Germany, was interested in our research and requested advice as to the molecular techniques to be carried out for the identification of this parasitic species. The studies carried out in collaboration with the German pharmaceutical company Or. Falk Pharma GMBH, allowed us

30 set up an identity test for T. suis eggs based on PCR techniques (Cutillas and coL, 2009). In this work, a review of the differential characteristics used up to that moment to distinguish both species is made.

Currently, the specific differentiation and diagnosis of trichuriasis is based on the detection of eggs in feces according to their morphological characteristics. This is a double problem: on the one hand, the eggs of the different species of Trichuris are practically identical, and on the other, that the number of eggs in feces is very low and, in some cases the use of combined techniques is recommended to avoid The false negatives. These problems are solved with the use of specific molecular markers that allow the identification of eggs in feces in a specific way. Until now there are specific kits on the market to carry out the extraction of AON from stool samples, but this invention provides, the procedure, the primers and the specific restriction enzymes necessary to carry out the specific diagnosis.

EXPLANATION OF THE INVENTION

By way of explanation of the invention ~ Use of restriction enzymes as molecular markers for the diagnosis of human trichuriasis and other trichuriasis of animate origin it is necessary to highlight how direct diagnoses based on the morphological characteristics of Trichuris eggs do not allow Make a specific diagnosis. The morphological variability of the eggs observed in the same species, the possibility of zoonosis: trichuriasis transmitted by dogs and pigs, climatic conditions and population migration, means that currently, epidemiological studies carried out by the different world agencies cannot be 100% reliable and, therefore, adequate measures cannot be taken to treat, control and prevent trichuriasis.

In addition, the proposed invention represents an advance in the control of wastewater, since the content of parasitic helminth eggs, as an indicator of the sanitary risk associated with the reuse of reclaimed water for irrigation without restrictions, is one of the quality parameters included in the third edition of the World Health Organization (WHO) guidelines published in 2006 under the original title of "Guidelines for the safe use of wastewater, excreta and greywater". WHO recommends a limit equal to less than 1 egg of parasitic helminths per liter of water for the safe reuse of an unrestricted irrigation water, and a more restrictive limit, equal to less than 0.1 egg of parasitic helminths per liter of water, when people under 15 can be exposed to contact with these waters.

RO 1620/2007 establishing the legal regime for the reuse of

S purified water in Spain collects these WHO recommendations and sets a maximum allowable value of 1 egg of intestinal nematodes in 10 liters of water for various options for urban, agricultural, industrial and environmental use of reclaimed water. These limits are associated with a surveillance protocol that includes, in most cases, a biweekly frequency of parasitological analyzes of the regenerated water, being

10 species of the genus Trichuris among the most commonly detected in Spanish wastewater. The eggs of the parasite that causes human trichuriasis, Trichuris trichiura, are morphologically almost identical to those of Trichuris suis and are considered by the World Health Organization (WHO) as one of the three preferred bioindicators to assess the quality of a regenerated water. Hence the importance that

15 acquires a good and reliable specific determination.

Based on the problem indicated, the proposed invention consists of a specific procedure based on molecular markers that will allow us to differentiate which species of Trichuris is infecting man, or which species of Trichuris is present in

20 any sample: feces, water, or others, that is, these molecular markers make the diagnosis feasible based on the deployment of the following procedure:

1. Collection and detection of the material under study (feces or water eggs).

25 2. Cultivation at 32 ° C of these eggs (Optional depending on the parasite load).

3.

DNA extraction from eggs extracted with the commercial kit Dneasy Blood & Tissue Kit (Qiagen).

Four.

Amplification of the AON with specific primers for the cytochrome oxidase 1 gene of the Trichuris species.

30 5. Purification of the PCR product obtained.

6. Digestion with specific restriction enzymes (endonucleases) that differentiate us the different species of Trichuris.

DESCRIPTION OF THE DRAWINGS

To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, according to a preferred example of practical implementation thereof, a set of figures is attached as an integral part of said description. where, for illustrative and non-limiting purposes, the following has been represented:

FIGURE 1.- Scheme of the amplification and purification procedure of the gene of the kycochrome oxidase 1 from eggs of T richuris spp.

FIGURE 2.-Sequences obtained for cytochrome oxidase 1 of the different Trichuris species. These sequences have been deposited in GenBank and are protected until publication.

FIGURE 3.-Scheme of the specific cut-off points of the different restriction enzymes in the sequences of the cytochrome oxidase 1 gene of the different Trichuris species.

FIGURE 4.- Characteristic restriction map of Hae 111 endonuclease for the different Trichuris species responsible for parasitism in man. MIX: molecular marker; T.t: Trichuris lrichiura; (H.s): Oven sapiens; C.g.k .: C% bus guerezae kikuyensis; Ts .: Trichuris suis; Tv .: Trichuris vulpis.

FIGURE 5.-Characteristic restriction map of the Mse I endonuclease for the different Trichuris species. MIX: molecular marker; Tt: Trichuris trichiura; (H.s): Oven sapiens; C.g.k .: C% bus guerezae kikuyensis; T.s .: Trichuris suis; T.v .: Trichuris vu / pis. T.m .: Trichuris muris; TA .: Trichuris aNico / ae; Tsk .: Trichuris skrjabini; Td .: Trichuris discolor, T.o .: Trichuris avis; EL: molecular marker.

EXAMPLE OF REALIZATION

Thus, by means of the invention "Use of restriction enzymes as molecular markers for the diagnosis of human Trichuriasis and other trichuriasis of animal origin" it is proposed to use a method based on the use of molecular markers for the specific differentiation of the Trichuris genus.

L. Most of the works published in recent years have been directed to the perfection and application of molecular techniques to the diagnosis and epidemiology of parasitic diseases. Thus, molecular techniques are not complicated and offer enormous advantages over conventional techniques in their application to the systematic study of parasites (Prichard, 1997) since: DNA is more stable than proteins; Nucleic acids have a special ability to hybridize with another simple chain of similar nucleic acid, offering a wide range of advantages. The Polymerase Chain Reaction (PCR) allows the possibility of amplifying multiple specific sequences of DNA or RNA. The Polymerase Chain Reaction (PCR) (Saiki et al., 1988) is a widely used method, since it allows the analysis of genes with minimal amounts of parasitic material (Gasser et al., 1993; Campbell et al., 1995), and does not require radioactive elements. Among the most commonly used PCR-based methods are: PCR-restriction fragments (PCR-RFLP).

The technique known as PCR-RFLP, is a method that detects the minimum variation in a gene when a single base substitution occurs, if it has led to the creation of a site capable of being digested by a restriction enzyme, or It has caused the disappearance of it. Restriction Endonucleases are enzymes of bacterial origin capable of cutting or digesting the double AON chain, recognizing a specific sequence of between 4 and 7 base pairs. In this technique, PCR is used to amplify a region of the gene. The PCR product is subjected to digestion by one or several restriction endonucleases. These digested products are visualized after agarose gel electrophoresis, ethidium bromide tefid, under UV light, being observed

the pattern of characteristic bands. This method requires previously knowing the portion of the AON to study in order to compare the patterns obtained.

A detail of the procedure, as an example to deploy for the realization of the technique and that has led us to the results that we propose to patent, is listed below:

1. Collection of the material under study

Once the faecal samples arrive at the laboratory in tightly closed bowls, the microscopic examination, directly and after concentration, of the feces for the visualization of Trichuris eggs is carried out. The concentration technique used is that of flotation in a solution of supersaturated sodium chloride (Schnieder et al., 1999, Cutillas et al., 2009).

Isolated eggs, if scarce, can be grown in a tube at 32 ° C for 3 to 4 weeks in 0.2% potassium dichromate solution (Horii and Usui, 1985).

2 . Egg amplification and sequencing of Trichuris spp. The molecular study of these parasitic nematodes comprises several stages:

2.1. Extraction of DNA:

The DNA extraction technique is based on the protocol of the Dneasy Blood & Tissue Kit commercial kit (Qiagen). Once the DNA is extracted, an aliquot in an 0.8% agarose gel and ethidium bromide is electrophoresed for visualization.

2.2. Amplification of the cox 1 gene of mtDNA (Figure 1) Amplification

The extracted DNA is subjected to an amplification reaction of the AON fragment under study following the following protocol:

Components Volume Final concentration

5 PCR buffer 10x S ~ I 1x 10mM 2 ~ I 0.4mM dNTP mix each CI, M9 SO mM 3 ~ I 1.SmM Primers () .lM each) 5) .ll 1 mM each DNA sample 5 III

10 Taq DNA Polymerase SU / ~ I) O.S f'1 2.5 Units Distilled water e.s.p. SO ~ I

The primers used for partial amplification of the cytochrome gene (15 oxidase subunit 1 (cox1) of the AONmt have been specifically designed from those described by Folmer et al. (1994) and Nagano et al (1999):

HC02198F S · -TGAIIIIIIGGTCACCCTGAAGTTTA-3 · comocebadordirecloy CORAS · -ACYACATAGTAGGTRTCATG-3 · as reverse 20 This reaction mixture is subjected to the following temperature cycles:

1. One cycle at 94 ° C for 5 minutes

2. 40 cycles consisting of: 94 oC for 60 seconds 48 oC for 60 seconds 72 oC for 60 seconds

3. One cycle at 72 oC for 7 minutes.

Upon completion of the peR reaction, the sample is subjected to a 2% agarose gel electrophoresis in TBE buffer at constant voltage, in the presence of elidia bromide and using a molecular weight marker. Subsequently, the gel is observed in a transilluminator, being able to visualize the band corresponding to the AON fragment under study. Finally, it is photographed with the KODAK 10 (Scientific imaging systems).

2.3. Purification and sequencing

The corresponding PCR product band is extracted from the agarose gel and purified following the protocol of the commercial kit of QIAEX 11 Extraction Kit (Qiagen). The amplified AON fragments are sequenced.

3. Study of the sequences obtained from mitochondrial DNA (Figure 2).

It has been studied:

3.1. Intraspecific variability: Which is based on knowing the differences in sequences (variability) between individuals of the same species. Once the pattern sequence or consensus is known.

3.2. Interspecific variability: The sequences obtained for T richuris lrichiura, T. suis and T vulpis, responsible for parasitisms in man and other species of this genus are studied comparatively with the possible sequences deposited in the GenBank to study the similarity between species of the genus Trichuris and other species of parasitic nematodes.

Four . Comparative study of the data obtained with other species of parasitic nematodes. Obtaining specific molecular markers (Figure 3, 4 and 5).

This phase of the study has consisted of using programs such as elustal W to carry

carried out the relevant alignments and similarity and homology calculations between

different species Likewise, the sequences obtained have been studied through

GenBank's "map" program to define specific restriction enzymes that differentiate us from the Trichuris species studied.

The results obtained can be seen in the table shown below:

Enzyme

T. suis T. vulpis T. trieltiura (Oven sapiens) T. trichiura (Papio, p.) T. lrichiura (Colobas guereza kikuyensis) T. orvicolae T. muris T. ovu T. discolor T. skrjabini

EcoRI

- 230 - - - - - - - -

Mse 1

85, 244, 324, 348 - 75,234 84,243 252 84 51 84, 243, 299 - 92,307

Hae lJI

- - 87 96 - - 63 156, 345 103 -

Honey

- - 327 336 - 171 138 99 106, 178 -

Pl'U TI

- - 246 255 - - - 255 - -

Ava l

193 - - - 201 - - - 199 200

Taq l

194 - - - 202 - - 54, 186 200 -

Xho l

193 - - - 201 - - - 199 -

Pst 1

- 213 - - - 211 - - - -

Dral

86 - 76 - - 85 52 85 - 93

Bc ll

- - - - - - - - - 227

BshT I

- - - - 359 - - - - 244

Ahll

- - - - - - - - - 393

BspJ9 1

- - - - - 336 - - - -

Blrbi

- 373 - - 380 - - 37 1 - -

Avr 11

- 138 - - - - - - - -

Acc III

- - - - - - - 314 - -

Aal)

- - - - - - - 3. 4. 5 - -

10 Thus, knowledge of these restriction enzymes allows us to specify the methodology for the specific identification of Trichuris eggs in three steps:

one.

Detection, extraction and amplification of DNA from Trichuris eggs using primers specific for the enzyme cox 1.

2.

Purification of amplified DNA

5 3. Digestion with specific restriction enzymes.

It is not considered necessary to make this description more extensive for any expert

in the field understand the scope of the invention and the advantages thereof

drift The techniques used and their application will be subject to variation whenever

10 And when this does not imply an alteration in the essentiality of the invention.

Claims (9)

1.-In vitro diagnostic method of human Trichuriasis and others of animal origin based on the use of restriction enzymes as molecular markers characterized by comprising the following steps: a) Isolation of Trichuris eggs from human feces, sewage. 10 b) Cultivation at 32 ° C of these eggs (optional depending on the parasite load). e) Extraction of AON from eggs and amplification by the PCR technique (polymerase chain reaction) of the cytochrome oxidase 1 gene. D) Purification and digestion of the PCR product with specific enzymes. 2.-In vitro diagnostic method of human Trichuriasis and others of animal origin according to claim 1 wherein the restriction enzymes used are (endonucleases) Hae 111, Mfe I and Pvu 11 that specifically cut for T richuris trichiura parasite of the 20 man but not cut in T. suis or T. vulpis. 3.-In vitro diagnostic method of human Trichuriasis and others of animal origin according to claim one wherein the restriction enzymes used are A vr 11 and Eco R I that specifically cut into the dog's parasite T. vulpis and responsible for zoonosis in the 25 man, but not in T suis or in T trichiura. 4.- In vitro diagnostic method of human Trichuriasis and others of animal origin according to First claim wherein the restriction enzymes used are Ava 1, Taq 1, Xho I that specifically cut in Trichuris suis pig parasite and zoonotic species to 30 man, but they do not cut in T vulpis or in T lrichiura parasite of man. 5.-In vitro diagnostic method of human Trichuriasis and others of animal origin according to claim one wherein the restriction enzyme used is the Mse I that differentiates specifically Trichuris suis, T. vu / pis, T. lrichiura, T. arvico / ae, T. muris, T. avis, T. discolor and T. skrjabini. 6.-In vitro diagnostic method of human Trichuriasis and others of animal origin according to claim 5 wherein the restriction enzyme used is the Ora I that specifically cuts for Trichuris trichiura parasite of man and T. suis but not in T. vu / pee. 7.-Restriction enzymes as molecular markers for the diagnosis of Trichuriasis according to claims 1, 2, 3, 4,5 and 6 characterized by the sequences of the 10 ci / oeroma oxidase 1 from which specific endonucleases have been extracted, and comprising the sequences SEO ID No. 1, SEO ID No. 2, SEO ID No. 3, SEO ID No. 4, SEO ID No. 5 , SE ID N '6, SE ID N' 7, SE ID N '8, SE ID N' 9 AND SE ID N'10. 8.-Use of the in vitro diagnostic method of human Trichuriasis and others of animal origin 15 according to claim one to amplify and sequence the cytochrome oxidase 1 gene of mitochondrial DNA. 9.-Use of restriction enzymes as molecular markers for the diagnosis of human Trichuriasis and other Trichuriasis of animal origin, according to claims 1, 2, 3, 4, 20 5, 6, 7 and 8, characterized by its primers designed to amplify the cytochrome oxidase 1 gene in all species of the genus Trichuris; HC02198F as direct primer and CORA as reverse, and comprising the sequences SEO ID No. 11 and SEO ID No. 12, respectively.