# Patent

## Title
Method for the detection of bacterial species of the genera anaplasma/ehrlichia and bartonella

## Abstract

The present invention relates to a method for the detection and identification of bacterial species belonging to the genera Anaplasma/Ehrlichia and Bartonella, and also provides triggers and probes required for its application, as well as associated kits.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 11/954,708, filed Dec. 12, 2007, which claims the benefit of priority to Spanish Patent Application No. ES 200701830, filed Jun. 29, 2007, both of which are hereby incorporated by reference in their entireties.


This application claims the benefit of priority to Spanish Patent Application No. ES 200701830, filed Jun. 29, 2007, which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to a method for the detection and identification of bacterial species belonging to the genera Anaplasma/Ehrlichia and Bartonella (ES 2264642), and also provides triggers and probes required for its application, as well as associated kits.
BACKGROUND
To date, about 200 zoonotic diseases (e.g., bartonelosis, leptospirosis, Lyme borreliosis, etc.), which affect humans and represent one of the main causes of death and entail substantial economic loss in third world countries, have been described. Coexistence with animals, lack of sanitary infrastructure and low cultural level continue to be the main allies of these diseases.
In the same manner, certain types of zoonosis which are widespread in third world countries are now thriving in industrialized countries as a consequence of population increases in urban and periurban areas, and increased movement of animals across international borders. These circumstances, amongst others, entail the risk of introducing exotic diseases into the environment.
Additionally, the frequent findings of arthropods infected by more than one of the pathogens included in the present invention, increases the possibility of more than one zoonotic disease being transmitted in a single sting. As a result, hospitalizations due to medical profiles produced by contact with animals or arthropods, such as mosquitoes, ticks, fleas, lice, mites, etc., which act as vectors or pathogen reservoirs, is becoming increasingly common. Said medical profiles, due to their high degree of similarity, do not allow a fast and reliable identification of the pathogenous agent, so that specific and fast treatment is not possible and is occasionally administered too late. This undoubtedly justifies the need for a comprehensive method to detect and identify bacterial species that cause zoonosis.
To date, the molecular diagnosis methods available are basically limited to the detection of pathogens based on antibody technology. This type of analysis, generally retrospective and with low sensitivity levels, is normally of little use to treating diseases in acute-phase states.
Another alternative for the detection and identification of pathogens is based on the application of culture mediums. These types of techniques are scarcely applicable to certain species of genera, such as Bartonella and Anaplasma/Ehrlichia, due to the fact that said species do not normally grow in regular culture mediums and may even require cellular cultures. As a result, these methodologies are isolated from regular practice in hospital microbiology laboratories. One of the most effective alternatives to these types of methodologies is the direct analysis of genetic material, based on Polymerase Chain Reaction (PCR) technology. Said technology, while being highly effective, is greatly limited by the difficulty in finding specific markers or regions, in addition to triggers and probes, which ensure reliable sample analysis.
A paper has recently been published (Blaskovic D. et al. 2005. FEMS Microbiology Letters 243:273-8) which describes a method based on ribosomal DNA analysis, although it uses universal triggers which amplify the genetic material of both target and non-target bacteria, due to which its sensitivity is substantially reduced.
Other methods, such as those described by U.S. Pat. Nos. 6,300,072 and 6,518,020, are capable of detecting and identifying bacteria of the genus Bartonella by using the same region (16S-23S), even though the number of species within this genus has increased substantially since said patents were filed and their approximation, which consists of discriminating between species according to the size of the amplicon obtained during PCR, is not useful for certain known species within the same genus which are similar in size to the amplified fragment.
SUMMARY OF THE INVENTION
The present invention relates to the use of genes 16S and msp2 and intergenic space 16S-23S for the detection and identification of the different bacterial species and groups of species that cause zoonosis, improving the previously described procedures based on its capacity to specifically detect a substantial number of bacterial species using probes and triggers with high sensitivity levels.
Therefore, the present invention solves the problem of the tediousness and complexity of detecting a high number of bacteria that cause zoonosis which can be clinically and/or epidemiologically indistinguishable, through the development of a detection method and Kit based on PCR technology. Specifically, the invention allows the analysis of different regions of bacterial DNA belonging to the genera Anaplasma/Ehrlichia and Bartonella in order to identify both genus and species, as shown in the following table (Table 1).
In most cases, the species identified correspond to cultured species and, in others, correspond to species isolated for the first time (non-cultured species). Said species have been obtained from samples of Meles meles (badger), Ixodes ricinus and Apodemus sylvaticus species and have been characterized by the sequences AJ269792 and SEQ ID NO:44-46, as shown in Table 3.









TABLE 1






BACTERIAL SPECIES
GENE








Anaplasma/Ehrlichia




A. phagocytophilum msp2
msp2



Ehrlichia ruminantium
16S



E. sennetsu
16S



E. risticii
16S



E. muris
16S



A. platys
16S



E. canis/E. ovina
16S



Bartonella




Generic
16S



B. talpae
16S-23S



Bartonella sp. *
16S-23S



B. phoceensis
16S-23S



B. rattimasiliensis
16S-23S



Bartonella sp. detected in Apodemus sylvaticus
16S-23S



B. rochalimae
16S-23S



Bartonella sp. detected in badger
16S-23S



Bartonella sp ** detected in Ixodes ricinus
16S-23S



Bartonella sp detected in Ixodes ricinus
16S-23S





* B. chomeli/schoenbuchensis/capreoli/birtlesii









BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. This figure represents two hybridization membranes for the validation of triggers and probes used to detect the Bartonella genus. In the membrane on the left, the absence of crossed reactivity between the different probes within the genus is shown. In the membrane on the right, the absence of crossed reactivity in samples which do not contain the Bartonella genus is shown. The S-CI2 probe refers to the Internal Amplification Control (IAC) probe.
FIG. 2. This figure represents two hybridization membranes for the validation of triggers and probes used to detect the genera Anaplasma/Ehrlichia. In the membrane on the left, the absence of crossed reactivity between the different probes within the genus is shown. In the membrane on the right, the absence of crossed reactivity in samples which do not contain the genera Anaplasma/Ehrlichia is shown. The S-CI2 probe refers to the IAC probe.



DETAILED DESCRIPTION OF THE INVENTION
According to a first aspect of the invention, said invention relates to a method (hereinafter, method of the invention) for the detection and identification, preferably simultaneous, of any of the bacterial species and genera, as indicated in Tables 2 and 3, comprising the following steps:

    
    
        a. Amplifying any of the sequences related to the group comprised of SEQ ID NO:1-17, 47 and/or their complementary sequences using specific triggers.
        b. Detecting the amplification of the sequences mentioned in step a), said amplification being indicative of the presence or absence of the bacterial genera or species that cause zoonosis, as indicated in Tables 2 and 3.
    
    


The triggers required to apply the method of the invention may be designed by means of multiple alignment with the sequences comprising SEQ ID NO:1-17 and 47 using computer programs such as CLUSTAL X, and allow the identification of highly preserved regions that will act as a mould for trigger design, which must subsequently be validated empirically.
According to a preferred embodiment of this aspect of the invention, the triggers are capable of hybridizing with different nucleotide regions of the genes 16S, msp2 and intergenic space 16S-23S (Tables 2 and 3), although the sequences of said triggers shall preferably be selected from the SEQ ID NO:18-25 group and/or their complementary sequences, these being capable of amplifying SEQ ID NO:1-17, 47 and/or their complementary sequences, in a preferably simultaneous manner. These triggers, in addition to simplifying the method, have the advantage of low or null reactivity with respect to samples of other species (see Table 5).
According to an even more preferred embodiment of this aspect of the invention, the detection of sequences SEQ ID NO:1-17, 47 and/or their complementary sequences may be carried out based on well-known methodologies within the art, preferably using probes. According to an even more preferred embodiment, said probes are capable of hybridizing between the positions of genes 16S, msp2 and intergenic space 16S-23S, as indicated in Tables 2 and 3, although said probes will preferably comprise the sequences selected from the group that comprises SEQ ID NO:26-42, 48 and/or their complementary sequences.
A second aspect of the invention relates to triggers capable of amplifying the sequences selected from the group comprising SEQ ID NO:1-17, 47 and their complementary sequences. Preferably, said triggers shall be capable of hybridizing between the nucleotide positions of genes 16S, msp2 and intergenic space 16S-23S, as indicated in Tables 2 and 3 (column 3). According to an even more preferred embodiment, the triggers comprise the sequences selected from the group SEQ ID NO:18-25 and/or their complementary sequences. Hereinafter, these will be referred to as triggers of the invention.
A third aspect of the invention relates to probes capable of specifically detecting any of the bacterial species and genera, as indicated in Tables 2 and 3 (column 6), said probes being capable of hybridizing between nucleotide positions of genes 16S, msp2 and intergenic space 16S-23S, as indicated in tables 2 and 3. According to an even more preferred embodiment, the probes have sequences selected from the group SEQ ID NO:26-47, 48 and/or their complementary sequences. Hereinafter, these will be referred to as probes of the invention.
A fourth aspect of the invention relates to an analysis kit for the identification of any of the bacterial genera or species, as indicated in tables 2 and 3, where said kit comprises any of the triggers or probes of the invention. Additionally, this kit may include all the reactive agents, buffers, supports, etc. required for its development, without limitation.







TABLE 2







Detection and identification of species belonging to the genera


Anaplasma/Ehrlichia












ORGANISM







Anaplasma







(Ehrlichia)
GENE
TRIGGER
PROBES
SEQUENCES
POSITION





A.
msp2
SEQ ID NO: 18
MSP2
SEQ ID NO: 1
EF143812


phagocytophilum

(EF143812 (1-22))
(SEQ ID

(223-243)




SEQ ID NO: 19
NO: 26)






(EF143812 (313-







334)





 


Generic
16S
SEQ ID NO: 20.
AEGEN
SEQ ID NO: 47
U02521




(U02521 (9-30)
(SEQ ID

(38-57)




SEQ ID NO: 21
NO: 48)






(U02521 (109-86)





 


Ehrlichia
16S
SEQ ID NO: 21
S-RUM
SEQ ID NO: 2
DQ640401


ruminantium

SEQ ID NO: 21
(SEQ ID

(23-45)





NO: 27)




 


E. sennetsu
16S
SEQ ID NO: 20.
S-SEN
SEQ ID NO: 3
M73225




SEQ ID NO: 21
(SEQ ID

(46-63)





NO: 28)




 


E. risticii
16S
SEQ ID NO: 20.
S-RIS
SEQ ID NO: 4
AY005439




SEQ ID NO: 21
(SEQ ID

(46-65)





NO: 29)




 


E. muris
16S
SEQ ID NO: 20.
S-MUR
SEQ ID NO: 5
AY587608




SEQ ID NO: 21
(SEQ ID

(16-37)





NO: 30)




 


A. platys
16S
SEQ ID NO: 20.
S-PLA
SEQ ID NO: 6
EF139459




SEQ ID NO: 21
(SEQ ID

(53-75)





NO: 31)




 


E. canis/
16S
SEQ ID NO: 20.
S-
SEQ ID NO: 7
EF011111


E. ovina

SEQ ID NO: 21
CANOVIN

(14-37)





(SEQ ID







NO: 32)
















TABLE 3







Detection and identification of species belonging to the genus


Bartonella












ORGANISM







Bartonella
GENE
TRIGGER
PROBES
SEQUENCES
POSITION





Generic
16S
SEQ ID NO: 22
BARTGEN2
SEQ ID NO: 8
AJ223780




(AJ223780
(SEQ ID

(1054-1075)




(961-979))
NO: 33)






SEQ ID NO: 23







(AJ223780







(1376-1398))





 


B. talpae
16S-
SEQ ID NO: 24
S-TOPO
SEQ ID NO: 9
SEQ ID NO:



23S
(AY116638
(SEQ ID

43 (90-113)




(455-478))
NO: 34)






SEQ ID NO: 25







(724-743







(AJ269786))





 


B. phoceensis
16S-
SEQ ID NO: 24
S-PHO
SEQ ID NO: 10
AY515123



23S
SEQ ID NO: 25
(SEQ ID

(659-679)





NO: 35)




 


B.
16S-
SEQ ID NO: 24
S-RAT
SEQ ID NO: 11
AY515122


rattimasiliensis
23S
SEQ ID NO: 25
(SEQ ID

(807-827)





NO: 36)




 


B. rochalimae
16S-
SEQ ID NO: 24
S-ROC
SEQ ID NO: 12
AF415211



23S
SEQ ID NO: 25
(SEQ ID

(414-434)





NO: 37)




 


Bartonella sp. *
16S-
SEQ ID NO: 24
CHOSCA
SEQ ID NO: 13
AY116639



23S
SEQ ID NO: 25
(SEQ ID

(438-461)





NO: 38)




 


Bartonella sp. in
16S-
SEQ ID NO: 24
S-APO38
SEQ ID NO: 14
AJ269792


Apodemus
23S
SEQ ID NO: 25
(SEQ ID

(425-445)


sylvaticus


NO: 39)




 


Bartobella sp in
16S-
SEQ ID NO: 24
S-TEJ
SEQ ID NO: 15
SEQ ID


Meles meles
23S
SEQ ID NO: 25
(SEQ ID

NO: 44





NO: 40)

(329-350)


 


Bartonella sp. in
16S-
SEQ ID NO: 24
S-G-13
SEQ ID NO: 16
SEQ ID


Ixodes ricinus 13
23S
SEQ ID NO: 25
(SEQ ID

NO: 45





NO: 41)

(92-111)


 


Bartonella sp. in
16S-
SEQ ID NO: 24
S-G-41
SEQ ID NO: 17
SEQ ID


Ixodes ricinus 41
23S
SEQ ID NO: 25
(SEQ ID

NO: 46





NO: 42)

(86-104)





* B. chomeli/schoenbuchensis/capreoli/birtlesii






Brief explanation of Tables 2 and 3:

    
    
        Column 1 (organism) indicates the bacterial species or group of species detected in each case. The Bartonella sp. group refers to a group of species belonging to this genus with a high degree of similarity and which are jointly detected through a method of the invention.
        Column 2 (gene) indicates the gene or genome region used to detect the bacterial species or group of species listed in column 1.
        Column 3 (trigger) indicates the sequence of the pair of triggers required to amplify the variable regions of the gene or intergenic space indicated in each table (column 2), in addition to the sequence in which they hybridize.
        Column 4 (probe) indicates the sequence of the probes used to detect the bacterial species or groups of species listed in column 1 of each table.
        Column 5 (sequence 5′-3′) indicates the references of the sequences of the variable regions which are amplified for the detection of each bacterial species or group of species.
        Column 6 indicates the sequence code of a gene region or genome region listed in column 2, in addition to the specific position of each sequence in which the probe indicated in column 4 hybridizes.
    
    


DETAILED DESCRIPTION
The present invention has allowed the development of an analysis method for the detection and identification of different bacterial genera and species using PCR or Multiple PCR technology. Methodology development required the analysis of intergenic space 16S-23S rRNA of genes 16S and msp2. These regions were analyzed combining different software applications and by comparison in databases, until the candidate regions susceptible to being used to apply the method were detected.
Said candidate regions were used to create a large number of triggers and probes, most of which, approximately 90%, were rejected after hybridization testing, until those which did not develop crossed reactivity with samples of different origin (FIGS. 1 and 2, Table 5) and, additionally, had high sensitivity levels, were finally selected.
Below is a detailed description of the materials and methods used in the development of the present invention, in addition to representative examples thereof. These examples do not limit the invention, but rather illustrate it, demonstrating the efficiency of the method of the invention. The use of these and other examples anywhere in the specification is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified form. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and can be made without departing from its spirit and scope. The invention is therefore to be limited only by the terms of the claims, along with the full scope of equivalents to which the claims are entitled.
EXAMPLES
Amplification, Hybridization, and Validation
This step includes the experimental analysis of the variable regions detected earlier using PCR for their validation. The isolated DNA was amplified using PCR, applying the following temperature cycle table and reaction mixture composition, together with the specific triggers used previously for said purpose.










Temperature Cycles









Temperature (° C.)
Time
Cycles 25





94
9′
1


94
15′′ 
 


60
1′
40 


65
4′
 


65
7′
1









PCR reaction

mixture composition for a final volume of 50 μL:














H2O:
According to final DNA volume


Buffer Taq Gold LD:
9 μL


Cl2Mg [3 mM]:
6 μL


dNTPs [200 mM]:
1 μL × 4


BSA [0.8 ug/uL]:
4 μL


14 specific Triggers (SEQ ID 1-2,
0.5 μL of each (7 μL)


SEQ ID 7-8, SEQ ID 25-26, SEQ ID



28-29, SEQ ID 31-32, SEQ ID 36-37,



SEQ ID 52-53) [50 pm/μL]:



Taq Gold LD:
0.5 μL [2.5 units]


Problem DNA:
maximum 800 ng









The amplicons were sequenced for their validation, verifying that the amplified sequence coincided with the variable sequences inferred from bioinformatic studies.
Subsequently, the amplicons were hybridized with specific probes according to the Reverse Line Blotting (RLB) protocol described by Sjoerd G. T. Rijpkema et al., Journal of Clinical Microbiology, December 1995, p. 3091-3095, although applying the following modifications (FIGS. 1 and 2):















Substrate:
Super Signal West Dura (Pierce, Ref: 34075)



Probes:
used with a concentration of between 0.2 and




3.2 picomoles/microliter



Incubation:
at 55° C.



Lavages:
at 52° C.









Hybridization results are shown in FIGS. 1 and 2, where it is shown that each of the probes of the invention become joined specifically to each of the amplicons of the bacterial species detected using a method of the invention.
Preparation of Samples and Multiple PCR
One of the advantages of using PCR and RLB technology-based identification systems is that pure bacterial cultures are not required. In this manner and upon validation of the triggers and probes using DNA samples of the different species and subspecies listed in Tables 2 and 3, a Multiple PCR-based analysis of a DNA control mixture (FIGS. 1 and 2) prepared under laboratory conditions was carried out, followed by a RLB test, using the specifically designed triggers and probes and the previously indicated temperature cycles and reaction mixture composition.
Detection of PCR Inhibitors
An IAC, which was amplified together with target DNA, was created for the detection of PCR inhibitors, using specific triggers (Table 4) designed according to the preserved regions of the AB183705 sequence belonging to the THC synthase gene of the Cannabis sativa species. Specifically, the IAC amplicon corresponds to a sequence of 371 pairs of bases, for which a probe was also designed for detection during RLB.







TABLE 4







IAC. Amplified gene, sequence of each of the triggers and


probes used in the process and their relative position.

















POSITION


ORGANISM
GENE
TRIGGER
PROBES
SEQUENCE
5′-3′





c. sativa
THC Syn-
SEQ ID NO:


AB183705



thase
51 (Cl-F)


(77-99)




SEQ ID NO:


AB183705




52 (Cl-R)


447-427


 


C. sativa
THC Syn-
SEQ ID 51
SEQ ID
SEQ ID
AB183705



thase
(Cl-F)
NO: 50
NO: 49
(281-302)




SEQ ID NO:
(S-Cl2)






52 (Cl-R)









Specificity of the Method

The high specificity of this method is based on the design and selection of the triggers and probes used, which were tested with another series of organisms (Table 5), following the previously described method, verifying that the formation of amplicons (FIGS. 1 and 2, right membrane) was in no case unspecifically detected.







TABLE 5







Specificity: unrelated bacterial, arthropod and


mammal species used during method development.










SPECIES
RLB RESULT













Bacteria



1
Brucella melitensis
Negative


2
Chlamydia penumoniae
Negative


3
Chlamydia psittaci
Negative


4
Legionella pneumophila
Negative


5
Leptospira interrogans
Negative


6
Mycoplasma pneumoniae
Negative


7
Treponema pallidum
Negative


8
Orientia tsutsugamushi
Negative



Arthropods
Negative


9
Ixodes ricinus
Negative


10
Ripicephalus sanguineus
Negative



Mammals
Negative


11
Apodemus sylvaticus
Negative


12
Human
Negative









All references cited and/or discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.

## Claims

1. A method for detecting simultaneously bacterial species that cause zoonosis, belonging to the genera Anaplasma/Ehrlichia and Bartonella, comprising the following steps:
a. placing a DNA sample under analysis in contact with a reaction mixture containing specific primers that will amplify one or more sequences from the gene msp2 and fragment 16S for Anaplasma/Ehrlichia, and from fragment 16S and 16S-23S for Bartonella 
b. amplifying the sequences by means of polymerase chain reaction;
c. simultaneously detecting amplification products formed in the previous step by means of probes comprising SEQ ID NO: 26, SEQ ID NO: 28-30, SEQ ID NO: 32-33, and SEQ ID NO: 35-38, or their fully complementary sequences, used individually or in combination, said detection being indicative of the presence or absence of zoonosis-causing bacteria.

2. The method according to claim 1, further comprising the step of identifying the bacterial species corresponding to the amplification products.

3. The method of claim 2, wherein the steps of detecting and identifying are simultaneous.

4. A kit for detecting simultaneously bacterial species that cause zoonosis, belonging to the genera Anaplasma/Ehrlichia and Bartonella, comprising the probes SEQ ID NO: 26, SEQ ID NO: 28-30, SEQ ID NO: 32-33, and SEQ ID NO: 35-38, or their fully complementary sequences.