BACKGROUND
huanglongbing  or citrus greening is the most devastating disease of citrus, threatening the citrus industry worldwide, and leading to massive reduction in fruit production as well as death of infected trees  <cit> .
the causal agents of hlb are three closely related gram-negative, phloem-limited α-proteobacteria candidatus liberibacter species  <cit> .
the heat tolerant strain ca.
l. asiaticus  is the most widespread in asia as well as in the usa whereas ca.
l. americanus  is mostly limited to south america  <cit> .
ca.
l. africanus  is heat sensitive and localized to the african continent.
all the three liberibacter species are currently uncultured and are known to reside in the sieve tubes of the plant phloem  <cit>  or in the gut of the phloem-feeding psyllids  <cit> .
psyllids are the natural vectors in transmitting the bacteria between plants  <cit> .
the asian psyllid, diaphorina citri kuwayama  is responsible for transmitting las and lam in asia and america, while the african citrus psyllid, trioza erytreae del guercio , is the natural vector of laf in africa  <cit> .
the characteristic symptoms of the infected plants include the yellow shoots, foliar blotchy mottles, along with poor flowering and stunting  <cit> .
hlb also results in poorly colored, unpleasant tasting, reduced size fruit that shows staining of vascular columella and seed abortion  <cit> .
generally the fruit may remain partially green, for this reason hlb is also called citrus greening  <cit> .
chronically infected trees are sparsely foliated and display extensive twig or limb die-back and eventually die within three to five years  <cit> .
moreover, the disorders induced in diseased plants vary with cultivar, tree maturity, time of infection, stages of disease and other abiotic or biotic agents that affect the tree  <cit> .
hlb symptoms also share certain similarities to nutrient deficiency  <cit> , citrus stubborn disease caused by spiroplasma citri <cit>  and a hlb-like disease caused by a phytoplasma  <cit> .
early diagnosis and differentiation of las infections from those defects and agents mentioned above, is thus critical to reducing the spread and devastation of this disease locally and via international trade, as well as minimizing the economic impact of potential false positive diagnoses.
importantly, hlb and the asian citrus psyllid  are expanding to new citrus production areas.
currently, asian citrus psyllid has been found in florida, texas, california, arizona, hawaii, louisiana, georgia, and alabama in the usa, as well as in parts of south and central america, mexico, and the caribbean.
meanwhile, hlb has not only been identified in florida, louisiana, south carolina, louisiana, georgia, texas and california of the usa; it has also been discovered in cuba, belize, jamaica, mexico, and other countries in the caribbean  <cit> .
while hlb and d. citri have been found in different producing areas, the number of infected trees and the psyllid vector population vary dramatically among different regions.
thus, different strategies of management of hlb are recommended for different regions, according to the corresponding severity of hlb and occurrence of psyllid vectors.
currently, no efficient management strategy is available to control hlb.
for the recently las-infected citrus producing areas such as california, prevention and eradication of hlb are the most efficient and cost-effective approaches.
additionally, las infected trees are most often found to be asymptomatic during the early stage of infection.
thus, accurate early detection of las in citrus plants and psyllids is critical for enacting containment measures in non-endemic citrus producing areas.
for the citrus producing areas without hlb, such as the mediterranean region, accurate detection is critical for the success of quarantine measures against ca.
liberibacter.
methods such as biological indexing using graft, dodder transmission  <cit> , isothermal loop amplification   <cit> , electron microscopy  <cit> , dna probes  <cit> , enzyme-linked immunosorbent assays   <cit> , conventional pcr  <cit>  and quantitative real-time pcr   <cit>  are used for the diagnosis and confirmation of hlb.
although diagnostic tools like conventional pcr and lamp showed good sensitivity, they were not consistent in detection of las bacterium from infected plant and psyllid materials  <cit> .
the current hlb diagnostic detection mainly employs qrt-pcr based methods due to their sensitive and quantitative nature.
the initial qrt-pcr oligonucleotide primer sets for the detection of las, targeted rplkajl-rpobc operon   <cit> , 16s ribosomal rna gene    <cit> , eub338f/eub518r  <cit> , alf518f/ eub518r  <cit>  or species specific variable regions.
eub338f/eub518r primers are universal to eubacteria  <cit> , while alf518f/eub518r primers identify α-proteobacteria universally  <cit>  including las, therefore not specific.
furthermore, the primers based on the conserved 16s and β-operon regions are popular but nevertheless have been shown to pose a potential specificity issue, as both false negatives and false positives have been reported  <cit> .
therefore, efforts have been directed towards developing effective qrt-pcr primers that target other non-conserved sequences.
recent studies made use of intragenic repeat regions of the prophage sequence for the detection of las by qrt-pcr  <cit> .
however, the intragenic repeat regions of the prophage sequence were also identified in lam.
therefore, these primer pairs, hyvi/hyvii did not distinguish between las and lam, posing a specificity issue  <cit> .
consequently, primer pairs that specifically detect las and make clear distinction among other phylogenetically closely related bacteria are essential.
here we took a complimentary approach to identify the genes that are unique to las by a bioinformatic analysis with the goal of expanding the arsenal of tools for las detection.
the advancement in the genome sequencing of las  <cit>  provides an opportunity to design primers based on species specific sequences for the detection of las.
we designed the oligonucleotide primer pairs specific to the identified unique genic signatures.
we further validated their specificities and selectivity against closely related strains that demonstrated the application to las-infected tissues and insect vectors by a qrt-pcr.
RESULTS
recently, the whole genome sequences of las  <cit>  have been sequenced.
this allows for systematic screening of unique las genes in a genome-wide fashion.
the availability of the genome sequences of the closely related species lam  <cit> , l. crescens   <cit>  and ca.
l. solanacearum   <cit> , further effectively helps in identification of unique regions, by minimizing the cross-species reactions, thereby enhancing the diagnostic identification of las in a more distinct manner.
bioinformatic analysis
several high-throughput applications have been developed recently to design diagnostic primers using the whole genome sequence information including kpath, insignia, tofi, and topsi  <cit> .
among them, kpath, insignia, and topsi have the potential to be used for design of real-time pcr primers for qrt-pcr based assays for las, whereas tofi is used to design signatures for microarray-based assays.
these methods mentioned above can be basically categorized into alignment-free and alignment-based approaches.
the alignment-free approach uses both coding and non-coding regions of the genome and is useful for the genomes with less accurate sequence information, but generally result in high false positive rates as it does not involve pre-screening of the selected genomic loci for their discriminatory ability  <cit> .
the alignment-based approach involves pre-screening of the selected genomic loci for their discriminatory ability  <cit> .
this approach does not consider the genome annotation of genic and non-genic information, but rather aligns bigger regions of the genome, hence prone to lose shorter discriminatory sequence regions.
additionally, discriminatory ability of the selected regions are screened bioinformatically only on limited number of closely related species, which provide more opportunities for false positives.
we therefore took a complementary bioinformatics approach by pre-screening shorter genic regions against the nucleotide sequence database  at ncbi, to identify all the possible unique genic regions from the las genome.
the natural selection acts more strongly on genic region, hence use of discriminatory sequences in this region results in less false positives as the organisms are under selection pressure  <cit> .
additionally, pre-screening against the nt is more effective as it contains the largest pool of well-annotated nucleotide sequences from different organisms.
we envisioned that these two steps would result in more specific detection of target organism with less false positives, hence are included in our bioinformatics approach.
there are ~ <dig> genes assigned to the las genome.
therefore, manual searching of each of these sequences against the nt database using blast program  <cit>  is a laborious and time consuming procedure.
hence, we automated this sequence similarity search step by developing a standalone perl script .
this script performed the similarity searches for each of the las gene against the specified database with hard-coded parameters for the blast program.
further, manual analysis of the resulting blast search output files is also laborious and time consuming; we therefore, automated this step by developing a second perl script .
this script automatically parsed all the blast output files and returned the las sequences for which, no hits were found in other organisms.
we refer to these sequences as probable unique sequences, because there are nearly no identical sequences found in other organisms .
we performed the sequence similarity searches first by using stringent e-value of ≤  <dig> × 10- <dig> against nt database .
this search resulted in ~ <dig> sequences that are unique to las.
this set of sequences is relatively high to validate experimentally; therefore, to further reduce the number of unique sequences, we performed the second sequence similarity search with a relaxed e-value of ≤  <dig>  this search resulted in  <dig> unique sequences.
the e-value of ≤  <dig> excludes the sequences with even little similarity to other organisms.
therefore, the resulting  <dig> unique sequences are considered unique to las and constitute the promising candidates for qrt-pcr based detection .
we further searched the  <dig> unique sequences of las against the phylogenetically closely related lso, lam, and lcr.
because these organisms are closely related, we used the stringent e-value threshold of ≤  <dig> × 10- <dig> for this similarity search.
in order to achieve this e-value, the sequences need to be highly similar between the las, lso, lam, and lcr.
therefore, this close species filter procedure potentially eliminates all the las sequence targets that could lead to false positive results in qrt-pcr based molecular diagnostic assays.
consequently, we further eliminated four conserved sequences from the list of  <dig> unique sequences, resulting in a total of  <dig> potential sequence signatures.
we could not apply this close species filter step against laf genome as its genome is yet to be sequenced.
five  of the  <dig> unique gene sequences namely clibasia_ <dig>  clibasia_ <dig>  clibasia_ <dig>  clibasia_ <dig> and clibasia_ <dig> are in the prophage region of the las genome.
all these five unique sequences are located upstream of the genomic locus clibasia_ <dig> encoding a phage terminase.
there are possibly  <dig> genes that represent the complete prophage genome within the las genome  <cit> , of which  <dig> open reading frames  are upstream of the phage terminase, while the remaining  <dig> orfs are downstream.
the prophage genes clibasia_ <dig>  and clibasia_ <dig>  have been targeted in previous studies by both conventional as well as qrt-pcr based assays  <cit> .
we further analyzed the genomic orientation of the  <dig> unique genes.
this analysis revealed that  <dig>  of them are oriented on the sense strand, while the remaining  <dig>  were present on the anti-sense strand .
the sequence length of these unique genes ranged from  <dig> to 2595 base pairs  .
designing of las specific primers and experimental validation of the specificity and sensitivity of qrt-pcr assay to detect las
based on the genome sequence of las strain psy <dig>  we designed  <dig> qrt-pcr primer pairs that specifically target the  <dig> unique sequences identified in our bioinformatic analyses .
we designed the melting temperature  of each of these primers to range from 59°c to 65°c with an optimum of 62°c.
the gc content of the primers ranged from 35% to 65% with an optimum of 50%.
the pcr amplicon sizes for each primer set are between  <dig> to 185 bp .
in addition to the novel primers designed in this work, we also used a set of control primers that have been previously used in a qrt-pcr based detection of las.
these known primers include 16s rdna pairs specific to the three different candidatus liberibacter species   <cit> , β-operon   <cit> , intragenic repeats regions of the prophage sequence   <cit> , and the primer pair specific to the plant cytochrome oxidase  gene  <cit>  as a positive endogenous control.
we performed qrt-pcr assays to test the specificity of the designed primers using total dna extracted from las-infected citrus plants as a template.
to further validate the specificity of these primers, we also included total dna from the phylogenetically closely related species lam and laf in our test.
additionally, dna extracted from healthy citrus plant was used as a negative control, whereas water served as a no template control.
the results of qrt-pcr assays are listed in table  <dig>
t
value of the qrt-pcr
#
c
t
r
<dig>
value
†
†
†las-infected psyllids total dna was serially diluted spanning up to five logs and used as a template in the qrt-pcr assay.
r <dig> and slope were further calculated from a plot of ct values versus log dilution factor.
#qrt-pcr was conducted by using template dna samples of las, laf, lam, c1: colletotrichum acutatum kla- <dig>  c2: elsinoe fawcettii, c3: xanthomonas axonopodis pv.
citrumelo <dig>  c4: xanthomonas citri subsp.
citri aw, c5: xanthomonas citri subsp.
citri a*, c6: xanthomonas citri subsp.
citri  <dig>  the ct values are average of three replicates for each primer pair.
ud: undetected; nt: not tested.
most of our novel custom designed primer pairs targeting the unique gene sequences were indeed found to be highly specific to las, as assessed by qrt-pcr assays .
among the  <dig> primer pairs,  <dig> produced amplicons only when las-infected citrus plant dna was used as a template, with an average ct value ranged from  <dig>  to  <dig> .
two primer pairs, p <dig> and p <dig>  didn’t produce any amplicons under the standard conditions tested.
the other three primer pairs, p <dig>  p <dig> and p <dig>  produced amplicons when las or laf infected plant dna was used as a template, indicating p <dig>  p <dig> and p <dig> could be used to detect both las and laf.
we were unable to filter for cross-reactivity of p <dig>  p <dig> and p <dig> in the bioinformatic analysis, because the laf genome sequence is currently unavailable.
with the exception of these three primer sets that showed amplicons with laf template, none of the other primer sets produced any amplicons with dna of lam, laf, and healthy citrus or water as template, which further confirms the specificity of these primers to the las.
we further evaluated the specificity of these primer sets using dna templates from various citrus associated fungal and bacterial pathogens including colletotrichum acutatum kla- <dig>  elsinoe fawcettii, xanthomonas axonopodis pv.
citrumelo  <dig>  x. citri subsp.
citri strains  <dig>  aw, and a*.
only two primers sets, p <dig> and p <dig> showed unspecific amplification against template dna extracted from fungal pathogen c. acutatum kla- <dig> .
c. acutatum causes citrus blossom blight, post-bloom fruit drop and anthracnose symptoms that are phenotypically distinguishable from citrus hlb.
the p <dig> and p <dig> were not filtered by the bioinformatic analysis since c. acutatum genome sequence was unavailable in the database.
because of the complexity of the natural microbial community and the limited number of sequences available in the current nucleotide sequence database, it is impossible to completely filter out all the potential false positives bioinformatically.
however, false positives could be identified experimentally by combining the different sets of primer pairs by a consensus approach  <cit> .
we eliminated these two primer sets from further evaluation in this study.
the melting temperature analysis of the amplicons produced from our novel primer set with las as a template indicated that amplicons were of a single species.
this suggests that there is no off target amplification for our primer pairs on the las genome.
overall, the experimental validation of the  <dig> novel primer sets specific to unique targets revealed that  <dig>  of these targets are indeed specific to the las genome .
this demonstrates the significance of the bioinformatics strategy employed here for identifying the suitable target regions for the detection of the bacteria by qrt-pcr based methods.
these  <dig> novel primer pairs were selected for further characterization.
to test the sensitivity of our designed novel primers, serial dilutions of las-infected psyllid dna was used as a template in the qrt-pcr assay.
this serial dilution qrt-pcr assay indicated that most of our novel primer pairs were able to detect las up to  <dig> dilutions from the initial template dna concentration, which is comparable to that of the primer set targeting las 16s rdna .
however, lower sensitivity was observed in the case of primer pairs p <dig>  p <dig>  p <dig> and p <dig>  which were eliminated from further study.
the remaining  <dig> primer pairs were able to detect las up to  <dig> dilutions, with a correlation co-efficient  between the ct values and dilutions .
this demonstrates the high sensitivity of these  <dig> primers in the detection of las.
qrt-pcr detection of las from plant and psyllid dna samples isolated from diverse locations in usa and china
in order to further demonstrate the degree of applicability of the  <dig> primer pairs in the detection of las from infected biological material, we performed qrt-pcr on the various las-infected plant and psyllid dna samples.
considering the potential variation in nucleotide sequences of las isolates in different geographic locations that might affect our detection due to the potential nucleotides changes of the target unique genes, we collected las-infected plant dna samples as tabulated in table  <dig>  from not only usa, but also from china, where las was reported more than 100 years ago  <cit> .
we tested the  <dig> primer pairs on  <dig> las-infected plant dna samples.
of these  <dig>   <dig> were collected from different locations in florida, usa , and the remaining five were collected from different locations in china .
additionally, las-infected psyllid dna samples collected from five different locations in florida, usa, were also included in the qrt-pcr assays .
t
value of qrt-pcr using infected plant dna samples as template
#
#las-infected plant dna samples were collected from  <dig> different locations in florida, usa, and  <dig> different locations in china.
the ct values indicated are average of three replicates for each primer pairs.
ud: under determined.
t
value of qrt-pcr using infected psyllid dna samples as template
#
#las-infected psyllid dna samples were collected from  <dig> different locations in florida, usa.
the ct values indicated are average of three replicates for each primer pair.
all the  <dig> primer pairs detected las from all  <dig> florida hlb diseased plant samples  and  <dig> psyllid dna samples  in a qrt-pcr assay, which further validated the detection applicability of our novel primers .
however,  <dig> of the  <dig> primer pairs  failed to produce amplicons with the infected plant dna sample from jiangxi and guangdong province, china .
primer pair p <dig> produced no amplicon with jiangxi sample, and produced unspecific amplicon with the guangdong sample .
interestingly, all these  <dig> primer pairs target the genes located in prophage region of the las genome .
these primers  based on prophage genes could detect las from florida, but not from jiangxi and guangdong province, china.
this is consistent with previous report  <cit> , that prophage was detected in only  <dig> % of the  <dig> hlb diseased citrus samples acquired in guangdong province, china, but was detected in  <dig> % of the  <dig> las positive citrus samples acquired in yunnan province, china.
this suggests that those prophage genes are not universally present in all strains of las.
alternately, the prophage sequences were found to be highly variable among the strains tested.
CONCLUSIONS
we have successfully designed  <dig> novel primer pairs, which are specific to las.
these primers will provide an additional arsenal to qrt-pcr based detection of las-infected plants and psyllids.
compared to the commonly used primers based on 16s rdna and β-operon, the  <dig> primers developed in this study have comparable sensitivity.
moreover, these primers could successfully differentiate las from lam, laf and other common microbes associated with citrus.

 huanglongbing or citrus greening is the most devastating disease of citrus, threatening the citrus industry worldwide, and leading to massive reduction in fruit production as well as death of infected trees. the causal agents of hlb are three closely related gram-negative, phloem-limited α-proteobacteria candidatus liberibacter species. the heat tolerant strain ca. l. asiaticus is the most widespread in asia as well as in the usa whereas ca. l. americanus is mostly limited to south america. ca. l. africanus is heat sensitive and localized to the african continent. all the three liberibacter species are currently uncultured and are known to reside in the sieve tubes of the plant phloem or in the gut of the phloem-feeding psyllids. psyllids are the natural vectors in transmitting the bacteria between plants. the asian psyllid, diaphorina citri kuwayama is responsible for transmitting las and lam in asia and america, while the african citrus psyllid, trioza erytreae del guercio, is the natural vector of laf in africa. the characteristic symptoms of the infected plants include the yellow shoots, foliar blotchy mottles, along with poor flowering and stunting. hlb also results in poorly colored, unpleasant tasting, reduced size fruit that shows staining of vascular columella and seed abortion. generally the fruit may remain partially green, for this reason hlb is also called citrus greening. chronically infected trees are sparsely foliated and display extensive twig or limb die-back and eventually die within three to five years. moreover, the disorders induced in diseased plants vary with cultivar, tree maturity, time of infection, stages of disease and other abiotic or biotic agents that affect the tree. hlb symptoms also share certain similarities to nutrient deficiency, citrus stubborn disease caused by spiroplasma citri and a hlb-like disease caused by a phytoplasma. early diagnosis and differentiation of las infections from those defects and agents mentioned above, is thus critical to reducing the spread and devastation of this disease locally and via international trade, as well as minimizing the economic impact of potential false positive diagnoses. importantly, hlb and the asian citrus psyllid are expanding to new citrus production areas. currently, asian citrus psyllid has been found in florida, texas, california, arizona, hawaii, louisiana, georgia, and alabama in the usa, as well as in parts of south and central america, mexico, and the caribbean. meanwhile, hlb has not only been identified in florida, louisiana, south carolina, louisiana, georgia, texas and california of the usa; it has also been discovered in cuba, belize, jamaica, mexico, and other countries in the caribbean. while hlb and d. citri have been found in different producing areas, the number of infected trees and the psyllid vector population vary dramatically among different regions. thus, different strategies of management of hlb are recommended for different regions, according to the corresponding severity of hlb and occurrence of psyllid vectors. currently, no efficient management strategy is available to control hlb. for the recently las-infected citrus producing areas such as california, prevention and eradication of hlb are the most efficient and cost-effective approaches. additionally, las infected trees are most often found to be asymptomatic during the early stage of infection. thus, accurate early detection of las in citrus plants and psyllids is critical for enacting containment measures in non-endemic citrus producing areas. for the citrus producing areas without hlb, such as the mediterranean region, accurate detection is critical for the success of quarantine measures against ca. liberibacter. methods such as biological indexing using graft, dodder transmission, isothermal loop amplification, electron microscopy, dna probes, enzyme-linked immunosorbent assays, conventional pcr and quantitative real-time pcr are used for the diagnosis and confirmation of hlb. although diagnostic tools like conventional pcr and lamp showed good sensitivity, they were not consistent in detection of las bacterium from infected plant and psyllid materials. the current hlb diagnostic detection mainly employs qrt-pcr based methods due to their sensitive and quantitative nature. the initial qrt-pcr oligonucleotide primer sets for the detection of las, targeted rplkajl-rpobc operon, 16s ribosomal rna gene, eub338f/eub518r, alf518f/ eub518r or species specific variable regions. eub338f/eub518r primers are universal to eubacteria, while alf518f/eub518r primers identify α-proteobacteria universally including las, therefore not specific. furthermore, the primers based on the conserved 16s and β-operon regions are popular but nevertheless have been shown to pose a potential specificity issue, as both false negatives and false positives have been reported. therefore, efforts have been directed towards developing effective qrt-pcr primers that target other non-conserved sequences. recent studies made use of intragenic repeat regions of the prophage sequence for the detection of las by qrt-pcr. however, the intragenic repeat regions of the prophage sequence were also identified in lam. therefore, these primer pairs, hyvi/hyvii did not distinguish between las and lam, posing a specificity issue. consequently, primer pairs that specifically detect las and make clear distinction among other phylogenetically closely related bacteria are essential. here we took a complimentary approach to identify the genes that are unique to las by a bioinformatic analysis with the goal of expanding the arsenal of tools for las detection. the advancement in the genome sequencing of las provides an opportunity to design primers based on species specific sequences for the detection of las. we designed the oligonucleotide primer pairs specific to the identified unique genic signatures. we further validated their specificities and selectivity against closely related strains that demonstrated the application to las-infected tissues and insect vectors by a qrt-pcr.  recently, the whole genome sequences of las have been sequenced. this allows for systematic screening of unique las genes in a genome-wide fashion. the availability of the genome sequences of the closely related species lam, l. crescens and ca. l. solanacearum, further effectively helps in identification of unique regions, by minimizing the cross-species reactions, thereby enhancing the diagnostic identification of las in a more distinct manner. bioinformatic analysis several high-throughput applications have been developed recently to design diagnostic primers using the whole genome sequence information including kpath, insignia, tofi, and topsi. among them, kpath, insignia, and topsi have the potential to be used for design of real-time pcr primers for qrt-pcr based assays for las, whereas tofi is used to design signatures for microarray-based assays. these methods mentioned above can be basically categorized into alignment-free and alignment-based approaches. the alignment-free approach uses both coding and non-coding regions of the genome and is useful for the genomes with less accurate sequence information, but generally result in high false positive rates as it does not involve pre-screening of the selected genomic loci for their discriminatory ability. the alignment-based approach involves pre-screening of the selected genomic loci for their discriminatory ability. this approach does not consider the genome annotation of genic and non-genic information, but rather aligns bigger regions of the genome, hence prone to lose shorter discriminatory sequence regions. additionally, discriminatory ability of the selected regions are screened bioinformatically only on limited number of closely related species, which provide more opportunities for false positives. we therefore took a complementary bioinformatics approach by pre-screening shorter genic regions against the nucleotide sequence database at ncbi, to identify all the possible unique genic regions from the las genome. the natural selection acts more strongly on genic region, hence use of discriminatory sequences in this region results in less false positives as the organisms are under selection pressure. additionally, pre-screening against the nt is more effective as it contains the largest pool of well-annotated nucleotide sequences from different organisms. we envisioned that these two steps would result in more specific detection of target organism with less false positives, hence are included in our bioinformatics approach. there are ~ genes assigned to the las genome. therefore, manual searching of each of these sequences against the nt database using blast program is a laborious and time consuming procedure. hence, we automated this sequence similarity search step by developing a standalone perl script. this script performed the similarity searches for each of the las gene against the specified database with hard-coded parameters for the blast program. further, manual analysis of the resulting blast search output files is also laborious and time consuming; we therefore, automated this step by developing a second perl script. this script automatically parsed all the blast output files and returned the las sequences for which, no hits were found in other organisms. we refer to these sequences as probable unique sequences, because there are nearly no identical sequences found in other organisms. we performed the sequence similarity searches first by using stringent e-value of ≤  × 10- against nt database. this search resulted in ~ sequences that are unique to las. this set of sequences is relatively high to validate experimentally; therefore, to further reduce the number of unique sequences, we performed the second sequence similarity search with a relaxed e-value of ≤  this search resulted in unique sequences. the e-value of ≤  excludes the sequences with even little similarity to other organisms. therefore, the resulting unique sequences are considered unique to las and constitute the promising candidates for qrt-pcr based detection. we further searched the unique sequences of las against the phylogenetically closely related lso, lam, and lcr. because these organisms are closely related, we used the stringent e-value threshold of ≤  × 10- for this similarity search. in order to achieve this e-value, the sequences need to be highly similar between the las, lso, lam, and lcr. therefore, this close species filter procedure potentially eliminates all the las sequence targets that could lead to false positive results in qrt-pcr based molecular diagnostic assays. consequently, we further eliminated four conserved sequences from the list of unique sequences, resulting in a total of potential sequence signatures. we could not apply this close species filter step against laf genome as its genome is yet to be sequenced. five of the unique gene sequences namely clibasia_ clibasia_ clibasia_ clibasia_ and clibasia_ are in the prophage region of the las genome. all these five unique sequences are located upstream of the genomic locus clibasia_ encoding a phage terminase. there are possibly genes that represent the complete prophage genome within the las genome, of which open reading frames are upstream of the phage terminase, while the remaining orfs are downstream. the prophage genes clibasia_ and clibasia_ have been targeted in previous studies by both conventional as well as qrt-pcr based assays. we further analyzed the genomic orientation of the unique genes. this analysis revealed that of them are oriented on the sense strand, while the remaining were present on the anti-sense strand. the sequence length of these unique genes ranged from to 2595 base pairs. designing of las specific primers and experimental validation of the specificity and sensitivity of qrt-pcr assay to detect las based on the genome sequence of las strain psy we designed qrt-pcr primer pairs that specifically target the unique sequences identified in our bioinformatic analyses. we designed the melting temperature of each of these primers to range from 59°c to 65°c with an optimum of 62°c. the gc content of the primers ranged from 35% to 65% with an optimum of 50%. the pcr amplicon sizes for each primer set are between to 185 bp. in addition to the novel primers designed in this work, we also used a set of control primers that have been previously used in a qrt-pcr based detection of las. these known primers include 16s rdna pairs specific to the three different candidatus liberibacter species, β-operon, intragenic repeats regions of the prophage sequence, and the primer pair specific to the plant cytochrome oxidase gene as a positive endogenous control. we performed qrt-pcr assays to test the specificity of the designed primers using total dna extracted from las-infected citrus plants as a template. to further validate the specificity of these primers, we also included total dna from the phylogenetically closely related species lam and laf in our test. additionally, dna extracted from healthy citrus plant was used as a negative control, whereas water served as a no template control. the results of qrt-pcr assays are listed in table  t value of the qrt-pcr # c t r value † † †las-infected psyllids total dna was serially diluted spanning up to five logs and used as a template in the qrt-pcr assay. r and slope were further calculated from a plot of ct values versus log dilution factor. #qrt-pcr was conducted by using template dna samples of las, laf, lam, c1: colletotrichum acutatum kla- c2: elsinoe fawcettii, c3: xanthomonas axonopodis pv. citrumelo c4: xanthomonas citri subsp. citri aw, c5: xanthomonas citri subsp. citri a*, c6: xanthomonas citri subsp. citri the ct values are average of three replicates for each primer pair. ud: undetected; nt: not tested. most of our novel custom designed primer pairs targeting the unique gene sequences were indeed found to be highly specific to las, as assessed by qrt-pcr assays. among the primer pairs, produced amplicons only when las-infected citrus plant dna was used as a template, with an average ct value ranged from to. two primer pairs, p and p didn’t produce any amplicons under the standard conditions tested. the other three primer pairs, p p and p produced amplicons when las or laf infected plant dna was used as a template, indicating p p and p could be used to detect both las and laf. we were unable to filter for cross-reactivity of p p and p in the bioinformatic analysis, because the laf genome sequence is currently unavailable. with the exception of these three primer sets that showed amplicons with laf template, none of the other primer sets produced any amplicons with dna of lam, laf, and healthy citrus or water as template, which further confirms the specificity of these primers to the las. we further evaluated the specificity of these primer sets using dna templates from various citrus associated fungal and bacterial pathogens including colletotrichum acutatum kla- elsinoe fawcettii, xanthomonas axonopodis pv. citrumelo x. citri subsp. citri strains aw, and a*. only two primers sets, p and p showed unspecific amplification against template dna extracted from fungal pathogen c. acutatum kla-. c. acutatum causes citrus blossom blight, post-bloom fruit drop and anthracnose symptoms that are phenotypically distinguishable from citrus hlb. the p and p were not filtered by the bioinformatic analysis since c. acutatum genome sequence was unavailable in the database. because of the complexity of the natural microbial community and the limited number of sequences available in the current nucleotide sequence database, it is impossible to completely filter out all the potential false positives bioinformatically. however, false positives could be identified experimentally by combining the different sets of primer pairs by a consensus approach. we eliminated these two primer sets from further evaluation in this study. the melting temperature analysis of the amplicons produced from our novel primer set with las as a template indicated that amplicons were of a single species. this suggests that there is no off target amplification for our primer pairs on the las genome. overall, the experimental validation of the novel primer sets specific to unique targets revealed that of these targets are indeed specific to the las genome. this demonstrates the significance of the bioinformatics strategy employed here for identifying the suitable target regions for the detection of the bacteria by qrt-pcr based methods. these novel primer pairs were selected for further characterization. to test the sensitivity of our designed novel primers, serial dilutions of las-infected psyllid dna was used as a template in the qrt-pcr assay. this serial dilution qrt-pcr assay indicated that most of our novel primer pairs were able to detect las up to dilutions from the initial template dna concentration, which is comparable to that of the primer set targeting las 16s rdna. however, lower sensitivity was observed in the case of primer pairs p p p and p which were eliminated from further study. the remaining primer pairs were able to detect las up to dilutions, with a correlation co-efficient between the ct values and dilutions. this demonstrates the high sensitivity of these primers in the detection of las. qrt-pcr detection of las from plant and psyllid dna samples isolated from diverse locations in usa and china in order to further demonstrate the degree of applicability of the primer pairs in the detection of las from infected biological material, we performed qrt-pcr on the various las-infected plant and psyllid dna samples. considering the potential variation in nucleotide sequences of las isolates in different geographic locations that might affect our detection due to the potential nucleotides changes of the target unique genes, we collected las-infected plant dna samples as tabulated in table  from not only usa, but also from china, where las was reported more than 100 years ago. we tested the primer pairs on las-infected plant dna samples. of these were collected from different locations in florida, usa, and the remaining five were collected from different locations in china. additionally, las-infected psyllid dna samples collected from five different locations in florida, usa, were also included in the qrt-pcr assays. t value of qrt-pcr using infected plant dna samples as template # #las-infected plant dna samples were collected from different locations in florida, usa, and different locations in china. the ct values indicated are average of three replicates for each primer pairs. ud: under determined. t value of qrt-pcr using infected psyllid dna samples as template # #las-infected psyllid dna samples were collected from different locations in florida, usa. the ct values indicated are average of three replicates for each primer pair. all the primer pairs detected las from all florida hlb diseased plant samples and psyllid dna samples in a qrt-pcr assay, which further validated the detection applicability of our novel primers. however, of the primer pairs failed to produce amplicons with the infected plant dna sample from jiangxi and guangdong province, china. primer pair p produced no amplicon with jiangxi sample, and produced unspecific amplicon with the guangdong sample. interestingly, all these primer pairs target the genes located in prophage region of the las genome. these primers based on prophage genes could detect las from florida, but not from jiangxi and guangdong province, china. this is consistent with previous report, that prophage was detected in only % of the hlb diseased citrus samples acquired in guangdong province, china, but was detected in % of the las positive citrus samples acquired in yunnan province, china. this suggests that those prophage genes are not universally present in all strains of las. alternately, the prophage sequences were found to be highly variable among the strains tested.  we have successfully designed novel primer pairs, which are specific to las. these primers will provide an additional arsenal to qrt-pcr based detection of las-infected plants and psyllids. compared to the commonly used primers based on 16s rdna and β-operon, the primers developed in this study have comparable sensitivity. moreover, these primers could successfully differentiate las from lam, laf and other common microbes associated with citrus. 
the oligonucleotide primer pairs based on conserved genes or regions, which include 16s rdna and the β-operon, have been widely employed in the detection of las by qrt-pcr. furthermore, by designing the primer pair specific to the identified signatures, we showed that most of our primer sets are able to detect las from the infected plant and psyllid materials collected from the usa and china by qrt-pcr. overall, primer pairs of the are found to be highly specific to las with no cross reactivity to the closely related species ca. we took a complimentary approach by systematically screening the genes in a genome-wide fashion, to identify the unique signatures that are only present in las by an exhaustive sequence based similarity search against the nucleotide sequence database. quantitative real-time pcr based molecular diagnostic assays have been routinely used in the detection and diagnosis of las.
 huanglongbing or citrus greening is a devastating disease of citrus. the gram-negative bacterium candidatus liberibacter asiaticus belonging to the α-proteobacteria is responsible for hlb in north america as well as in asia. currently, there is no cure for this disease. early detection and quarantine of las-infected trees are important management strategies used to prevent hlb from invading hlb-free citrus producing regions. quantitative real-time pcr based molecular diagnostic assays have been routinely used in the detection and diagnosis of las. the oligonucleotide primer pairs based on conserved genes or regions, which include 16s rdna and the β-operon, have been widely employed in the detection of las by qrt-pcr. the availability of whole genome sequence of las now allows the design of primers beyond the conserved regions for the detection of las explicitly.  we took a complimentary approach by systematically screening the genes in a genome-wide fashion, to identify the unique signatures that are only present in las by an exhaustive sequence based similarity search against the nucleotide sequence database. our search resulted in probable unique signatures. furthermore, by designing the primer pair specific to the identified signatures, we showed that most of our primer sets are able to detect las from the infected plant and psyllid materials collected from the usa and china by qrt-pcr. overall, primer pairs of the are found to be highly specific to las with no cross reactivity to the closely related species ca. l. americanus and ca. l. africanus.  we have designed qrt-pcr primers based on las specific genes. among them, are suitable for the detection of las from las-infected plant and psyllid samples. the repertoire of primers that we have developed and characterized in this study enhanced the qrt-pcr based molecular diagnosis of hlb. detection systemdiagnosticcandidatus liberibacter asiaticusgreeninghuanglongbingbacteriapsyllidcitrus 