# Patent

## Title
Method and kit for the detection of bacterial species by means of DNA analysis

## Abstract

The present invention relates to the detection and identification of different bacterial species, all of which cause zoonosis, based on DNA analysis. More specifically, the invention provides the primers, probes, genes and genic regions required to apply a method for the simultaneous detection of bacteria and bacterial groups belonging to the genera Anaplasma, Ehrlichia, Borrelia, Bartonella, Coxiella, Rickettsia and Francisella based on Multiple PCR analysis by RLB (Reverse Line Blotting), in addition to providing a kit to carry out said analysis.

## Background
This application is a U.S. national phase application under 35 U.S.C. §371 of International Patent Application No. PCT/ES2006/070082 filed Jun. 15, 2006, which claims the benefit of priority to Spanish Patent Application No. 200501481 filed Jun. 17, 2005 and U.S. Provisional Patent Application No. 60/691,231 filed Jun. 17, 2005, all of which are hereby incorporated by reference in their entireties. The International Application was published in Spanish on Dec. 28, 2006 as WO 2006/136639.


FIELD OF THE INVENTION
The present invention relates to the detection and identification of different bacterial species, all of which cause zoonosis, based on DNA analysis. More specifically, the invention provides a method and kit for the simultaneous detection of bacteria and bacterial groups belonging to the genera Anaplasma, Ehrlichia, Borrelia, Bartonella, Coxiella, Rickettsia and Francisella based on DNA amplification.
BACKGROUND
To date, about 200 zoonotic diseases (bartonelosis, leptospirosis, Lyme borreliosis, etc.), which affect humans have been described. In third world countries, they represent one of the main causes of death and entail substantial economic loss. Coexistence with animals, lack of sanitary infrastructure and low cultural level continue to be the main allies of these diseases.
Certain types of zoonosis 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, which entails the risk of introducing exotic diseases into the environment.
These circumstances, coupled with the frequent findings of arthropods infected by more than one of the pathogens included in the present invention, increase the possibility of more than one of the bacterial species included in the present invention being transmitted in a single sting.
As a result, hospitalizations due to medical profiles produced by human contact with animals or different classes of 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 detection method.
The diagnostic methods currently available are limited to detecting antibodies which, in general, is retrospective and of little use to treating patients in acute-phase states. Culture is not considered a diagnostic method, due both to its technological complexity, which excludes it from regular practice in hospital microbiology laboratories, and to the need for P3 facilities.
Molecular diagnosis by genome amplification by means of PCR represents a diagnostic option of great value. However, clinical samples of sufficient quantity for pathogen testing or the methodology required to carry out different tests are not always available.
A paper has recently been published (Blaskovic D. et al. 2005. Oligo-based detection of tick-borne bacteria. FEMS Microbiology Letters 243:273-8) which describes a method for the detection of 5 out of 6 pathogens proposed by the present invention. Said method is based on ribosomal DNA analysis and uses universal primers, 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 DNA region (intergenic space 16S-23S). However, 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.
While the method provided by the present invention also proposes using intergenic region 16S-23S for the detection of species belonging to the genus Bartonella, improvements have been introduced with respect to the previously described procedures, as it is capable of detecting a much wider range of species within the same and other genera, using completely new probes and primers with maximum sensitivity levels.
For the detection of Coxiella burnetii, the present invention uses the same primers and DNA region (insertion sequence IS1111) as the previously described methods. Said detection has been improved by combining it with another series of completely new tests aimed at identifying other bacterial species, which can be transmitted by the same vectors and also provide a new hybridization probe for the detection of Coxiella burnetii. 
DETAILED DESCRIPTION
Definitions: Multiple PCR or Multiplex PCR: PCR (Polymerase Chain Reaction) is a system whereby the number of copies of a specific nucleotide sequence of an organism is amplified or increased using two primers. Multiple PCR or Multiplex PCR is a variation of PCR which allows the simultaneous amplification of more than one target sequence using more than one pair of primers.
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 method and Kit for the simultaneous detection of bacterial species that cause zoonosis belonging to the genera: Anaplasma, Ehrlichia, Borrelia, Bartonella, Coxiella, Rickettsia and Francisella. 
The solution found by the present invention includes simultaneously analyzing different bacterial DNA regions to determine which species are present in both cases. Specifically, the 16S rRNA gene is analyzed in order to detect the presence of Anaplasma, Ehrlichia and Borrelia; intergenic space 23S-5S rRNA is analyzed to detect the presence of Rickettsia; the gene which codes for the precursor of the main membrane protein TUL4 is analyzed to detect the presence of Francisella; the transposase IS1111 gene is analyzed to detect the presence of Coxiella and intergenic space 16S-23S is analyzed to detect the presence of Bartonella. 
According to the above, a first aspect of the invention relates to a method for the sample-based detection of bacteria, comprised of the following steps:

    
    
        i) Placing the sample under analysis in contact with a reaction mixture containing specific primers to carry out Multiplex PCR.
        ii) Amplifying by means of polymerase chain reaction.
        iii) Identifying the formation of the products in the previous step, said information being indicative of the presence or absence of zoonosis-causing bacteria.
    
    


In relation to this first aspect of the invention, said invention provides a method to simultaneously detect:

    
    
        Anaplasma phagocytophilum, A. bovis, A. equi, A. marginale, A. centrale and A. ovis. 
        Ehrlichia chaffeensis and E. Ewingii. 
        Bartonella henselae, B. quintana, B. clarridgeiae, B. elizabethae, B. grahamii, B. vinsonii subspecies berkhofii, B. vinsonii subspecies vinsonii, B. vinsonii subspecies aurupensis, B. bacilliformis, B. alsatica, B. bovis, B. doshiae, B. koehlerae, B. schoen-buchensis, B. taylori and B. tribocorum. 
        All of the species belonging to the genus Borrelia. 
        Coxiella burnetii. 
        Any subspecies of Francisella turalensis, including F tularensis subsp. tularensis, F. tularensis subsp. holarctica and F. tularensis subsp. novicida which are jointly detected, and variant 3523 of the same species and so-called endosymbionts of different species of ixodides and argasides, which are detected differentially.
        The genus Rickettsia, and group that causes spotted fever and the group that causes typhus, the species Rickettsia akari, R. bellii, R. slovaca, R. conorii, R. aeschlimannii, R. ricketsii, R. sibirica, R. helvetica, R. felis, R. australis, R. prowazekii and R. typhy (R. mooserii).

all of which are capable of causing zoonosis, jointly infecting an individual and being difficult to identify by simple observation of medical profiles, based on the amplification and analysis of the genes or specific genic regions presented in Tables 1-6.

    
    


According to a specific embodiment of this first aspect of the invention, DNA fragments included or comprised within the sequences, the access numbers of which are shown in Tables 1-6 below, are amplified.
According to a more specific embodiment of this first aspect of the invention, the amplified regions have a size of between 99 and 686 nucleotides and contain variable regions used for identification. According to an even more specific embodiment of the invention, the variable regions contain or are included within sequences SEQ ID NO:55 to SEQ ID NO:93 or complementary sequences, the positions of which are shown in Tables 1 to 6.
According to another embodiment of this first aspect of the invention, the amplification products that allow different bacterial species and groups to be identified are detected by means of probes. According to a more preferred embodiment, said probes have a length of between 15 and 25 nucleotides. And, according to an even more preferred embodiment, the probes have sequences which comprise or are included within sequences SEQ ID NO:3-6; SEQ ID NO:9-24; SEQ ID NO:27; SEQ ID NO:30; SEQ ID NO:33-35; SEQ ID NO:38-51; or complementary sequences (Tables 1-6).
The primers can be designed by means of multiple alignment using computer programs such as CLUSTAL X, which allows the identification of highly conserved regions that act as moulds. According to another specific embodiment of this first aspect of the invention, the primers hybridize the genes indicated in Tables 1 to 6 below and particularly those with sequences with the access numbers shown in Tables 1-6 below. According to an even more specific embodiment, the primers have sequences which comprise or are included within SEQ ID NO:1-2; SEQ ID NO:7-8; SEQ ID NO:25-26; SEQ ID NO:28-29; SEQ ID NO:31-32; SEQ ID NO:36-37; or complementary sequences.
Brief explanation of the Tables:

    
    
        Column 1 (organism) indicates the bacterial species or group of bacterial species detected in each case.
        Column 2 (gene) indicates the gene or genome region used to detect the bacterial species or group of species in column 1.
        Column 3 (primer) indicates the sequence of the pair of primers required to carry out the amplification of variable gene regions or genome regions indicated in each Table (column 2).
        Column 4 (probe) indicates the sequence of the probes used to detect the bacterial species or group of species referenced in column 1 of each Table.
        Column 5 (sequence 5′-3′) indicates the sequence references of the variable regions which are amplified to detect each bacterial species or group of species.
        Column 6 (position 5′-3′):
        
            The first row: indicates a sequence code relative to a gene or genome region referenced in column 2, in addition to the specific position of said sequence in which the primer is hybridized (column 3).
            The second to the last row of each Table indicate a sequence code relative to a gene or genome region referenced in column 2, in addition to the specific position of said sequence to which the probe is joined (column 4).
        
        
    
    









TABLE 1







Anaplasma and Ehrlichia: sequence of each of the primers and probes used


in the process and their relative position within gene 16S rRNA.












ORGANISM
GENE
PRIMER
PROBES
SEQUENCE 5′-3′
POSITION 5′-3′





Anaplasma spp
16S
SEQ ID 1


 9-30


Ehrlichia spp

(16S/AE-F)


(U02521)




SEQ ID 2


109-86 




(16S/AE-R)


(U02521)


Anaplasma
16S
SEQ ID 1
SEQ ID 3
SEQ ID 57
52-73


phagocytophilum

(16S/AE-F)
(S-PHA)

(U02521)


A. bovis

SEQ ID 2


 8-29


A. equi

(16S/AE-R)


(AF470698)







 8-29







(AF172167)


Ehrlichia
16S
SEQ ID 1
SEQ ID 4
SEQ ID 58
51-71


chaffeensis

(16S/AE-F)
(S-CHA)

(AF147752)




SEQ ID 2







(16S/AE-R)





E. ewingii
16S
SEQ ID 1
SEQ ID 5
SEQ ID 59
46-66




(16S/AE-F)
(S-EWI)

(U96436)




SEQ ID 2







(16S/AE-R)





A. marginale
16S
SEQ ID 1
SEQ ID NO 6
SEQ ID 60
53-71


A. centrale

(16S/AE-F)
(S-MCO)

(AJ633048)


A. ovis

SEQ ID 2


72-90




(16S/AE-R)


(AF414869)







72-90







(AF414870)
















TABLE 2







Bartonella: sequence of each of the primers and probes used in the process


and their relative position within intergenic space 16S-23S rRNA.












ORGANISM
GENE
PRIMER
PROBES
SEQUENCE 5′-3′
POSITION 5′-3′





Bartonella spp.
16S-23S
SEQ ID 7


494-515




(BAR/16-23F)


(AF369527)




SEQ ID 8


908-889




(BAR/16-23R)


(AF369527)


B. henselae
16S-23S
SEQ ID 7
SEQ ID 9
SEQ ID 61
793-814




(BAR/16-23F)
(S-HENS)

(AF369527)




SEQ ID 8







(BAR/16-23R)





B. quintana
16S-23S
SEQ ID 7
SEQ ID 10
SEQ ID 62
622-641




(BAR/16-23F)
(S-QUIN)

(AF368396)




SEQ ID 8







(BAR/16-23R





B. clarridgeiae
16S-23S
SEQ ID 7
SEQ ID 11
SEQ ID 63
512-531




(BAR/16-23F)
(S-CLAR)

(AF312497)




SEQ ID 8







(BAR/16-23R





B. elizabethae
16S-23S
SEQ ID 7
SEQ ID 12
SEQ ID 64
807-827




(BAR/16-23F)
(S-ELIZ)

(L35103)




SEQ ID 8







(BAR/16-23R





B. grahamii
16S-23S
SEQ ID 7
SEQ ID 13
SEQ ID 65
491-514




(BAR/16-23F)
(S-GRAH2)

(AJ269790)




SEQ ID 8







(BAR/16-23R





B. vinsonii berkhofii
16S-23S
SEQ ID 7
SEQ ID 14
SEQ ID 66
2242-2261




(BAR/16-23F)
(S-VIN-B)

(AF143446)




SEQ ID 8







(BAR/16-23R





B. vinsonii arupensis
16S-23S
SEQ ID 7
SEQ ID 15
SEQ ID 67
686-706




(BAR/16-23F)
(S-VIN-A1)

(AF442952)




SEQ ID 8







(BAR/16-23R





B. vinsonii vinsonii
16S-23S
SEQ ID 7
SEQ ID 16
SEQ ID 68
821-841




(BAR/16-23F)
(S-VIN-A2)

(AF312504)




SEQ ID 8







(BAR/16-23R





B. bacilliformis
16S-23S
SEQ ID 7
SEQ ID 17
SEQ ID 69
474-493




(BAR/16-23F)
(S-BACI)

(AJ422181)




SEQ ID 8







(BAR/16-23R





B. alsatica
16S-23S
SEQ ID 7
SEQ ID 18
SEQ ID 70
589-608




(BAR/16-23F)
(S-ALS)

(AF312506)




SEQ ID 8







(BAR/16-23R





B. bovis
16S-23S
SEQ ID 7
SEQ ID 19
SEQ ID 71
455-478




(BAR/16-23F)
(S-BOV2)

(AY116638)




SEQ ID 8







(BAR/16-23R





B. doshiae
16S-23S
SEQ ID 7
SEQ ID 20
SEQ ID 72
724-743




(BAR/16-23F)
(S-DOSH)

(AJ269786)




SEQ ID 8







(BAR/16-23R





B. koehlerae
16S-23S
SEQ ID 7
SEQ ID 21
SEQ ID 73
778-803




(BAR/16-23F)
(S-KOE)

(AF312490)




SEQ ID 8







(BAR/16-23R





B. schoenbuchensis
16S-23S
SEQ ID 7
SEQ ID 22
SEQ ID 74
446-466




(BAR/16-23F)
(S-SCHO2)

(AY116639)




SEQ ID 8







(BAR/16-23R





B. taylori
16S-23S
SEQ ID 7
SEQ ID 23
SEQ ID 75
655-673




(BAR/16-23F)
(S-TAY)

(AJ269784)




SEQ ID 8







(BAR/16-23R





B. tribocorum
16S-23S
SEQ ID 7
SEQ ID 24
SEQ ID 76
692-713




(BAR/16-23F)
(S-TRIB)

(AF312505)




SEQ ID 8







(BAR/16-23R
















TABLE 3







Borrelia: sequence of each of the primers and probes used in


the process and their relative position within gene 16S rRNA.












ORGANISM
GENE
PRIMER
PROBES
SEQUENCE 5′-3′
POSITION 5′-3′





Borrelia spp.
16S
SEQ ID 25


336-356




(BOF-3)


(AJ224139)




SEQ ID 26


567-547




(BOR)


(AJ224139)


Borrelia
16S
SEQ ID 25
SEQ ID 27
SEQ ID 77
364-383




(BOF-3)
(SG-BOR3)

(AJ224139)




SEQ ID 26







(BOR)
















TABLE 4







Coxiella: sequence of each of the primers and probes used in the process and


their relative position within insertion sequence (transposase) IS1111.












ORGANISM
GENE
PRIMER
PROBES
SEQUENCE 5′-3′
POSITION 5′-3′





Coxiella
Transposase
SEQ ID 28


200-221


burnetii
IS1111
(TRANS 1)


(M80806)




SEQ ID 29


885-865




(TRANS 2)


(M80806)


Coxiella
Transposase
SEQ ID 28
SEQ ID 30
SEQ ID 78
520-539


burnetii
IS1111
(TRANS 1)
(S-IS1111)

(M80806)




SEQ ID 29







(TRANS 2)
















TABLE 5







Francisella: Amplified gene, sequence of each of the primers


and probes used in the process and their relative position.












ORGANISM
GENE
PRIMER
PROBES
SEQUENCE 5′-3′
POSITION 5′-3′





Francisella
17 kDa
SEQ ID 31


593-617


spp.
Tul4
(FT594)


(M32059)




SEQ ID 32


825-804




(FT827)


(M32059)


F. tularensis
17 kDa
SEQ ID 31
SEQ ID 33
SEQ ID 79
658-680



Tul4
(FT594)
(S-TUL)

(M32059)




SEQ ID 32







FT827





Variant
17 kDa
SEQ ID 31
SEQ ID 34
SEQ ID 80
169-188


3523
Tul4
(FT594)
(S-TUL3523)

(AY243029)




SEQ ID 32







FT827





Endosymbionts
17 kDa
SEQ ID 31
SEQ ID 35
SEQ ID 81
533-553



Tul4
(FT594)
(S-ENDOS2)

(AY375423)




SEQ ID 32







(FT827)
















TABLE 6







Rickettsia: sequence of each of the primers and probes used in the process and their


relative position within genes 238, 58 rRNA and within intergenic space 23S-5S rRNA.












ORGANISM
GENE
PRIMER
PROBES
SEQUENCE 5′-3′
POSITION 5′-3′





Rickettsia
23S-5S
SEQ ID 36


 1-22


spp.

(RCK/23-5-F)


(AY125012)




SEQ ID 37


388-367




(RCK/23-5-R)


(AY125012)


Generic
23S-5S
SEQ ID 36
SEQ ID 38
SEQ ID 82
51-71




(RCK/23-5-F)
(SG-RICK)

(AY125012)




SEQ ID 37







(RCK/23-5-R)





Spotted Fever
23S-5S
SEQ ID 36
SEQ ID 39
SEQ ID 83
123-141


Group

(RCK/23-5-F)
(SG-SFG)

(AY125012)




SEQ ID 37







(RCK/23-5-R)





R. akari
23S-5S
SEQ ID 36
SEQ ID 40
SEQ ID 84
291.105-291.126




(RCK/23-5-F)
(S-AKA4)

(AAFE01000001)




SEQ ID 37







(RCK/23-5-R)





R. bellii
23S-5S
SEQ ID 36
SEQ ID 41
SEQ ID 85
2721-2743




(RCK/23-5-F)
(S-BELLII)

(U11015)




SEQ ID 37







(RCK/23-5-R)





R. slovaca
23S-5S
SEQ ID 36
SEQ ID 42
SEQ ID 86
194-211




(RCK/23-5-F)
(S-SLO)

(AY125009)




SEQ ID 37







(RCK/23-5-R)





R. conorii
23S-5S
SEQ ID 36
SEQ ID 43
SEQ ID 87
186-204




(RCK/23-5-F)
(S-CON)

(AY125012)




SEQ ID 37







(RCK/23-5-R)





R. aeschlimannii
23S-5S
SEQ ID 36
SEQ ID 44
SEQ ID 88
183-204




(RCK/23-5-F)
(S-AESCH)

(AY125016)




SEQ ID 37







(RCK/23-5-R)





R. rickettsii
23S-5S
SEQ ID 36
SEQ ID 45
SEQ ID 89
2814-2833


R. sibirica

(RCK/23-5-F)
(S-RI/SI)

(U11022)




SEQ ID 37







(RCK/23-5-R)





R. helvetica
23S-5S
SEQ ID 36
SEQ ID 46
SEQ ID 90
360-342




(RCK/23-5-F)
(S-HELV)

(AY125017)




SEQ ID 37







(RCK/23-5-R)





R. felis
23S-5S
SEQ ID 36
SEQ ID 47
SEQ ID 55
186-207




(RCK/23-5-F)
(S-FEL)

(SEQ ID 55)




SEQ ID 37







(RCK/23-5-R)





R. australis
23S-5S
SEQ ID 36
SEQ ID 48
SEQ ID 56
230-249




(RCK/23-5-F)
(S-AUS)






SEQ ID 37







(RCK/23-5-R)





Grupo Tifus
23S-5S
SEQ ID 36
SEQ ID 49
SEQ ID 91
2804-2827




(RCK/23-5-F)
(SG-TG)

(U11018)




SEQ ID 37







(RCK/23-5-R)





R. prowazekii
23S-5S
SEQ ID 36
SEQ ID 50
SEQ ID 92
2824-2846




(RCK/23-5-F)
(S-PROW)

(U11018)




SEQ ID 37







(RCK/23-5-R)





R. typhi
23S-5S
SEQ ID 36
SEQ ID 51
SEQ ID 93
188-211


(R. mooserii)

(RCK/23-5-F)
(S-TYPHI)

(AY125019)




SEQ ID 37







(RCK/23-5-R)









Given the abundance of PCR inhibitors, such as humic and fulvic acid, heavy metals, heparin, etc. which can produce false negatives and, despite the methods that exist to reduce the concentration of this type of molecules, we recommend (cf. J. Hoorfar et al., “Making internal Amplification control mandatory for diagnostic PCR” J. of Clinical Microbiology, December 2003, pp. 5835) that the PCR tests contain an Internal Amplification Control (IAC). Said IAC is no more than a DNA fragment which is amplified simultaneously with the target sample, in such a way that its absence at the end of the testing process indicates the presence of factors which have caused unwanted development of the PCR.
A second aspect of the invention relates to a method similar to that described in the first aspect of said invention, including at least one IAC, preferably comprised of a DNA sequence of the Tetrahydrocannabinol Synthase gene of the Cannabis sativa species and, more preferably, of a sequence with access number AB183705.
According to a more preferred embodiment, a region of the AB183705 sequence is amplified, said sequence being included within SEQ ID NO:94 or complementary sequences (Table 7).
According to another preferred embodiment, the region is amplified by means of specific primers, the sequences of which comprise or are included within SEQ ID NO:52 and SEQ ID NO:53 or complementary sequences.







TABLE 7







Internal control: Amplified gene, sequence of each of the primers


and probes used in the process and their relative position.












ORGANISM
GENE
PRIMER
PROBES
SEQUENCE 5′-3′
POSITION 5′-3′





IAC
THC
SEQ ID 52


77-99


(Cannabis
Synthase
(CI-F)


(AB183705)


sativa)

SEQ ID 53


447-427




(CI-R)


(AB183705)


Cannabis
THC
SEQ ID 52
SEQ ID 54
SEQ ID 94
281-302


sativa
Synthase
(CI-F)
(S-CI2)

(AB183705)




SEQ ID 53







(CI-R)









Within the context of this description, the term “specific” implies that the primers comprise a nucleotide sequence fully complementary to the genes or genic fragments used by the present invention.
The term “variable regions” refers to DNA sequences which allow for the identification of the bacterial species and groups identified by the present invention.
According to another embodiment of the second aspect of the invention, IAC amplification is detected by means of hybridization with probes. According to a more preferred embodiment, said probes have a length of 15 to 25 nucleotides. And, in an even more preferred embodiment, said probes have a sequence comprised or included in SEQ ID NO:54 or complementary sequences.
The method provided by the present invention allows for the detection of the aforementioned bacteria and bacterial groups, independent of sample origin. Said samples may be obtained from biopsies, scrapings, insects, biological fluids (blood, urine, saliva, etc.), field, etc. Once taken, the sample is pretreated in order to carry out Multiple PCR and subsequent amplicon identification.
The invention also provides diagnosis kits to apply the method described by the invention, which contain:

    
    
        Specific primers with sequences: SEQ ID 1-2, SEQ ID 7-8, SEQ ID 25-26, SEQ ID 28-29, SEQ ID 31-32, SEQ ID 36-37 and optionally SEQ ID 52 y 53 as IAC.
        Probes with sequences: SEQ ID 3-6, SEQ ID 9-24, SEQ ID 27, SEQ ID 30, SEQ ID 33-35, SEQ ID 38-51 and optionally SEQ ID 54 (S-Cl2) as IAC.
    
    


In the same way, said kits can include all the reactive agents required to apply any of the methods described. This includes, without any type of limitation, the use of buffers, polymerases, cofactors to optimize their activity, contamination-preventing agents, etc. On the other hand, the kits can include all of the supports and containers required for their startup and optimization.
The advantages of the present method and the kits with which to apply it include: speed (39 species and bacterial groups can be detected in less than 8 hours), specificity (the initiators used are specific to each species or bacterial group) and a high level of sensitivity.
Within the context of the specification description and claims, the word “comprises” and its variations, such as “comprising”, does not intend to exclude other additives, components, constituent elements or stages. Both the examples and complementary drawings do not intend to limit the invention, but should rather be considered an aid to better understand it.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Hybridization membrane showing the validation of primers, probes, and variable regions for the detection of Anaplasma and Ehrlichia (A), Borrelia (B), Francisella (C), Bartonella (D) and Coxiella (E) species, by means of specific probes (Tables 1-5). The S-Cl2 probe refers to the IAC probe (Table 7).
FIG. 2. A) Hybridization membrane showing the validation of primers, probes and variable regions for the detection of Rickettsia species; B) Hybridization membrane showing an example of simultaneous detection of species belonging to the 7 genera. In this example: A. phagocytophilum, A. marginale. E. chaffeensis, E. ewingi, B. henselae, B. burgdorferi, F. tularensis tularensis, R. conorii and R. prowazekii, tested at 103, 102 and 10 equivalent genome/copies. In both cases (A and B) the S-Cl2 probe, which refers to the IAC probe (Table 7), is used.
FIG. 3. Hybridization membrane showing the results of a specificity study carried out within the indicated group of probes (Tables 1-7) on different species of bacteria, arthropods and mammals. The results reveal that the probes are not joined to the samples of the organisms tested in any case. The S-Cl2 probe refers to the IAC probe (Table 7).



EXAMPLE
The present invention is next described by reference to an example. The use of this 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.
Alignments, Primer, and Probe Designs
Conserved regions were identified by comparing and aligning multiple sequences obtained from public databases such as Genbank, based on which specific primers were designed (Table 1-6) for use in Multiple PCR. The compatibility between primers, in addition to their optimal concentration, was empirically tested in the same manner as the magnesium salts and bovine serum albumin.
Variable regions were identified based on the selected sequence alignments, which allowed probes to be designed for the differentiation of bacterial species and genic groups (Tables 1-6) through RLB (Reverse Line Blotting) (Kaufhold A, Podbielski A, Baumgarten G, Blokpoel M, Top J, Schouls L. Rapad Typing of group-a streptococci by the use of DNA amplification and nonradioactive allele-specific oligonucleotide probes. FEMS Microbiology Letters 119: 19-25 (1994)).
A first specificity analysis of each of the probes was carried out by comparing its sequence against public databases (Genbank) using computer programs such as BLAST. Specificity was subsequently demonstrated by carrying out tests on a variety of DNA samples of different bacterial and eukaryotic species (FIG. 3).
DNA Culture Mediums and Isolation
The species and genic groups selected for identification were obtained from private collections: the Spanish Type Culture Collection (CECT) and/or sample bank available at the National Microbiology Center (CNM). All of the species analyzed are shown in Table 8.
The isolation of genetic material was carried out using well-known procedures within the art and available on the market (DNA Mini Kit, Qiagen, N. Reference: 51304).







TABLE 8







Origin of DNA used in the invention










NATIVE DNA



ORGANISM
(origin)
SYNTHETIC DNA *





Anaplasma phagocytophilum
X (1)



A. marginale
X (1)



Ehrlichia chaffeensis

X


E. ewingii

X


Borrelia burgdorferi
X (2)



B. garinii
X (2)



B. afzelii
X (2)



B. lusitaniae
X (2)



B. jap{acute over (o)}nica
X (2)



B. hermsii
X (2)



B. parkeri
X (2)



Francisella tularensis tularensis
X (3)



F. tularensis subesp. holarctica
X (3)



F. tularensis subesp. Novicida
X (3)



Francisella variant 3523

X


Francisella Endosimbiontes

X


Bartonella alsatica
X (4)



B. bacilliformis
X (4)



B. bovis
X (4)



B. clarridgeiae
X (4)



B. doshiae
X (4)



B. elizabethae
X (4)



B. grahamii
X (4)



B. henselae
X (4)



B. koehlerae
X (4)



B. quintana
X (4)



B. schoenbuchensis
X (4)



B. taylorii
X (4)



B. tribocorum
X (4)



B. vinsonii subesp. Arupensis
X (4)



B. vinsonii subesp. Berkhofii
X (4)



B. vinsonii subesp. Vinsonii
X (4)



Coxiella burnetii
X (5)



Rickettsia aeschlimannii

X


R. akari
X (5)



R. australis
X (5)



R. bellii
X (5)



R. conori
X (5)



R. felis
X (5)



R. Helvetica
X (5)



R. rickettsii
X (5)



R. sibirica

X


R. slovaca
X (5)



R. prowazekii

X


R. typhi
X (5)



Brucella melitensis
X (6)



Chlamydia pneumoniae
X (7)



C. psittaci
X (7)



Escherichia coli
X (6)



Legionella pneumophila
X (8)



Leptospira interrogans
X (4)



Micoplasma pneumoniae
X (7)



Ochrobactrum antropi
X (4)



Orientia tsutsugamushi
X (5)



Pseudomonas aeruginosa
X (4)



Salmonella enterica Typhi
X (4)



Streptococcus pneumoniae
X (6)



Treponema pallidum
X (7)



Ixodes ricinus
X (9)



Dermacentor marginatus
X (9)



Rhipicephalus sanguineus
X (9)



Apodemus sylvaticus
X (9)



Human DNA
 X (10)



Internal Control

X





Origin of Native DNA:


(1): Positive sample.


(2): Axenic culture medium, as described in: Benach J L, Coleman J L, and Golightly M G. 1988. A murine monoclonal antibody binds an antigenic determinant in outer surface protein A, an immunodominant basic protein of the Lyme disease spirochete. J. Immunol. 140: 265-72.


(3): Axenic culture medium, as described in: Anda P, Segura del Pozo J, Diaz Garcia J M, Escudero R, Garcia Peña F J, Lopez Velasco M C, Sellek R E, Jimenez Chillaron M R, Sanchez Serrano L P, Martinez Navarro J F. 2001. Waterborne outbreak of tularemia associated with crayfish fishing. Emerg. Infect. Dis. 7 (Suppl): 575-82.


(4): Composition of axenic culture mediums specific to each species available at: Jacomo et al. (2002) Clinical and Diagnostic Laboratory Immunology, 9 (1) 8-18.


(5): Propagation in cellular cultures using the “shell vial” technique, as described in: Marrero M, Raoult D. 1989. Centrifugation-shell vial technique for rapid detection of Mediterranean spotted fever rickettsia in blood culture. Am. J. Trop. Med. Hyg. 40: 197-9.


(6): “Mueller Hinton” agar culture enriched with 5% of ram blood.


(7): DNA extracted from slides for indirect commercial immunofluorescence.


(8): Axenic culture medium, as described in: Edelstein P H. 1981. Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental samples. J. Clin. Microbiol. 14: 298-303.


(9): DNA extracted from pathogen-free specimens.


(10): DNA extracted from clinical samples of patients with unrelated diseases.






Synthetic DNA

Synthetic DNA was prepared according to the corresponding sequences listed in Tables 1 to 7 (Column 6), by means of consecutive elongation of the DNA chain by PCR, using primers with approximately 70 nucleotides, of which approximately 20 nucleotides interoverlapped.
Amplification, Hybridization and Validation
This step included the experimental analysis of the variable regions detected earlier using PCR for their validation. The isolated DNA was amplified using PCR (Saiki et al., (1985) Science 230, 1350; 1354), applying the following temperature cycle table and reaction mixture composition, together with the specific primers used previously for said purpose.










Temperature Cycles









Temperature (° C.)
Time
Cycles












94
9′
1


94
15′′ 



60
1′
40


65
4′



65
7′
1









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 Primers (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 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 2A):















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.



Wash:
at 52° C.









Hybridization results are shown in FIGS. 1 and 2A, where it is shown that each of the probes of the invention become joined specifically to the amplicons of each of the bacterial species detected using the method of the invention.
Preparation of Samples and Multiple PCR
One of the advantages of using PCR and RLB analysis-based identification systems is that pure bacterial cultures are not required. In this manner and upon validation of the primers and probes using DNA samples of the different species and subspecies listed in Tables 1-6, prepared following the procedures listed in Table 8 and analyzed in duplicate, a Multiple PCR-based analysis of a DNA control mixture prepared under laboratory conditions was carried out, followed by the RLB test, using the specifically designed primers and probes and the previously indicated temperature cycles and reaction mixture composition, the results of which are shown in FIG. 2B. In said figure it is shown that it was possible to carry out the simultaneous detection of the bacterial species present in the sample.
Detection of PCR Inhibitors
An internal amplification control (IAC), which was amplified together with the target DNA, was created for the detection of PCR inhibitors, using specific primers (Table 7) designed according to the conserved regions of the AB183705 sequence (Table 7) 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 (Table 7) for detection during RLB analysis.
Specificity of the Method
The high specificity of this method is based on the specificity of the primers and their probes, which were tested with another series of organisms (Table 9), following the method described by the present invention, verifying that the formation of amplicons detectable by means of hybridization (FIG. 3) was not detected in any case.







TABLE 9







Specificity: unrelated species of bacteria, arthropods


and mammals used in method development










SPECIES
RLB RESULT












All All
Bacteria
All All


1
Brucella melitensis
Negative


2
Chlamydia pneumoniae
Negative


3
C. psittaci
Negative


4
Escherichia coli
Negative


5
Legionella pneumophila
Negative


6
Leptospira interrogans
Negative


7
Mycoplasma pneumoniae
Negative


8
Ochrobactrum antropi
Negative


9
Orientia tsutsugamushi
Negative


10
Pseudomonas aeruginosa
Negative


11
Salmonella enterica Typhi
Negative


12
Streptococcus pneumoniae
Negative


13
Treponema pallidum
Negative



Arthropods
Negative


14
Ixodes ricinus
Negative


15
Dermacentor marginatus
Negative


16
Rhipicephalus sanguineus
Negative



Mammals
Negative


17
Apodemus sylvaticus
Negative


18
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 the simultaneous detection of bacterial species that cause zoonosis belonging to the genera Anaplasma, Ehrlichia, Bartonella, Borrelia, Coxiella, Francisella and Rickettsia comprising the following steps:
(a) placing a sample for analysis in contact with a reaction mixture containing specific primers SEQ ID NOs: 1, 2, 7, 8, 25, 26, 28, 29, 31, 32, 36 and 37, to perform multiplex polymerase chain reaction;
(b) amplifying the sequences by means of polymerase chain reaction: and
(c) detecting and identifying the amplification products formed in the previous step, said detection information being indicative of the presence or absence of zoonosis-causing bacteria.

2. The method of claim 1, wherein the bacterial species are:
(a) Anaplasma phagocytophilum, A. Bovis, A. equi, A. marginale, A. centrale and A. ovis; 
(b) Ehrlichia chaffeensis and E. Ewingii; 
(c) Bartonella henselae, B. quintana, B. clarridgeiae, B. elizabethae, B. grahamii, B. vinsonii subspecies berkhofii, B. vinsonii subspecies vinsonii, B. vinsonii subspecies aurupensis, B. bacilliformis, B. alsatica, B. bovis, B. doshiae, B. koehlerae, B. schoenbuchensis, B. taylori and B. tribocorum; 
(d) species belonging to the genus Borrelia; 
(e) Coxiella burnetti; 
(f) any subspecies of Francisella turalensis, including F. tularensis subsp. tularensis, F. tularensis subsp. holartica and F. tularensis subsp. novicida which are jointly detected, and variant 3523 of the same species and so-called endosymbionts of different species ixodides and argasides, which are differentially detected; and
(g) species belonging to the genus Rickettsia, the group that causes spotted fever, and the group that causes typhus; and the species Rickettsia akari, R. bellii, R. slovaca, R. conorii, R. aeschilmannii, R. ricketsii, R. sibirica, R. helvetica, R. felis, R. australis, R. prowazekii, and R. typhy (r. mooserii).

3. The method of claim 1, wherein the simultaneous detection of the amplification products is carried out using one or more probes having a sequence selected from SEQ ID NOs: 3-6, 9-24, 27, 30, 33-35, and 38-51.

4. The method of claim 1, further comprising the use of at least one internal amplification control.

5. The method of claim 4, wherein the use of the internal amplification control comprises:
(a) amplifying SEQ ID NO: 94 with specific primers; and
(b) detecting the amplification product formed in the previous step.

6. The method of claim 5, wherein one or more of the specific primers has a sequence selected from SEQ ID NOs: 52 and 53.

7. The method of claim 4, wherein the amplification product detection is carried out using the probe with SEQ ID NO: 54.