Microb. Resour. Syst. 32
Dec.
（2）
：123
2016 ─ 131, 2016

Vol. 32, No. 2

A cocktail PCR and DNA strip method for quick confirmationof multiple pathogenic factors in BSL3 stock cultures
Takayuki Ezaki1)＊, Izumi Kanazawa1), Sayoko Hayashi3), Masahiro Hayashi2),
Ibrahim Eldesoky1) and Hajime Fukunaga1)
Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine,2)
Anaerobic Laboratory, Life Science Research Center, Gifu University
1-1 Yanagido, Gifu 501-1194, Japan3)
Gifu University of Medical Science, 795-1 Aza-Nagamine, Ichihiraga, Seki, Gifu 501-3892, Japan1)

Repositories of culture collections of Biosafety Level 3 (BSL3) pathogens are expected to provide information on thepathogenic factors present in stock bacteria. However, it is difficult to maintain valid pathogenic information on each of
the different BLS3 pathogens because quality control of the stock strains generally has to be performed by a few staff
members.
We developed a common platform to differentiate six different PCR amplicons on a DNA strip. The six amplicons canbe differentiated with the help of three position markers printed on the strip. With this method, the pathogenic factors
in BSL3 pathogens can be determined within 40 min after cultured bacteria are picked up from a single colony.

INTRODUCTION

The provision of quality information on specificpathogens associated with Japanese Infectious
Diseases (SPJID) through the activities of the
National Bioresource Project of the Japanese
Ministry of Education, Culture, Sports, Science andTechnology, is especially important for both the collection of cultures and their use. The pathogenic
factors of Biosafety Level 3 (BSL3) pathogens and
SPJID are often encoded on plasmids or insertion
sequences on the bacterial chromosomes. These factors are, therefore, unstable and are often“cured”(i.e. eliminated) from the host. The important pathogenic factor invE (virB) of Shigella spp. and enteroinvasive Escherichia coli is required for the invasiveness of these bacteria (Mitobe et al., 2009). Thisfactor is encoded on a plasmid and is easily cured
from the host when stock bacteria are cultured.
Once the factor has been eliminated from the host, it
is difficult to differentiate these pathogens from nonpathogenic E. coli. The Vi antigen of Salmonella
enterica serovar Typhi is the most important virulence factor used for differentiating this serovar
from the 2000 other serovars of S. enterica, but theCorresponding author＊

E-mail: ezaki2016@gmail.com
Accepted: February 26, 2016

Vi synthesis gene clusters are naturally deleted
from the host chromosome (Hashimoto et al., 1996,
Hashimoto & Khan, 1997).
Capsular antigen fraction 1 (caf1) (Galyov, 1990)
and plasminogen activator surface protease (pla) arelocated on plasmids of Yersinia pestis (Guinet &Carniel, 2012). Once the plasmids are cured fromthe host, it becomes very difficult to differentiate Y.
pestis from the closely related Yersinia pseudotuberculosis.
Type strains and strains used for serotyping of
SPJID are especially important for maintaining quality information on the pathogenic factors in thesebacteria. Simple and rapid confirmation methods
are required so that these strains can be checked
before they are distributed. To fulfill this need, wedeveloped a tagged-cocktail PCR method (Hayashi,2013) and established a protocol to differentiate the
PCR amplicons by using a DNA strip containing six
printed anti-tags and three position markers. We
applied this technology to confirm the presence of
pathogenic factors of BSL3 and SPJID strains. The
PCR amplification method was also optimized forthis with the use of a low-profile PCR tube and aquick amplification PCR machine. We established a
protocol to confirm the presence of pathogenic factors of BSL3 and SPJID strains within 40 min fromthe time bacteria were collected from a single colony on a culture plate.

─ 123 ─

Cocktail PCR and DNA strip for quick confirmation of pathogenic factors

MATERIALS AND METHODS

Cell cultures
Strains from the Gifu Type Culture Collection(GTC), and the Japan National Collection of Bacterial
Pathogens (JNBP), along with wild Y. pestis strains
stocked at the Mongolian National Center for
Zoonotic Diseases (Ulaanbaatar, Mongolia) wereused. The tagged primers used are listed in Table
1. Upstream primers were labeled with a common
polyA sequence and downstream primers werelabeled with six different tags. These primers weresynthesized by Sigma-Aldrich Japan (Tokyo, Japan)
and Tohoku Bio-Array Corporation (Sendai, Japan).
The DNA strips printed with six different anti-tags
and three position markers were kindly provided by
Tohoku Bio-Array Corporation (Fig. 1).
Examination methods
Bacteria from a single colony plated the day
before and incubated overnight were suspended in
500 ml of distilled water, and the solution was boiled
to disrupt the cells. A volume of 2.5 ml of the solution was used for cocktail PCR analysis. The concentrations of the single-colony suspensions ranged
from 107 to 108 cfu/ml. To determine the detection
limit, cell suspensions of 101 to 108 cfu/ml were also
prepared. For rapid PCR, a low-profile PCR tube
was selected and a total of 10 ml of the reaction mixtures was used for analysis. The reaction mixturescontained 2.5 ml of 4× primer mixture (0.8 mM), 2.5 mlof crude DNA suspension, and 5 ml of 2× premixed
Taq polymerase (TakaraBio, Kyoto, Japan). After
denaturation at 97℃ for 2 min, samples were amplified under the following two-step PCR conditions:
60℃ for 10 s and 97℃ for 5 s. Thirty PCR cycles
were performed. The total amplification time waswithin 30 min.
After PCR amplification, 10 ml of a 0.5% blue latex
suspension labeled with polyT was added to the
PCR tube, and a DNA strip was inserted into the
PCR tube for chromatography. Ten minutes later,
the blue latex had been completely absorbed by the
DNA strip and the target line that captured the
PCR amplicon on the DNA strip had turned blue.
Sensitivity of the DNA strip
Two-fold serial dilutions of the PCR products of E.
coli shiga2-positive strain 157:H7 (GTC 14509) wereprepared. PCR products in the diluted solutions
were detected with a standard latex solution to

Ezaki et al.

determine the endpoint of detection. Ten-fold serial
dilutions of a Salmonella enterica GTC 3P0637 cellsuspension from 108 to 101 cfu/ml were prepared,
and the invA amplicon was detected by using SYBR
green (Takara Bio, Kyoto, Japan) and a Step One
Plus PCR machine (Applied Bio, Tokyo, Japan).
After amplification, the amplicon was also detectedby using the DNA-strip method, and the sensitivitiesof the two methods were compared.
Screening for pathogenic factors
Bacteria from a single colony plated the day
before and incubated overnight were suspended in
500 ml of distilled water to prepare a 108 cfu/ml cellsuspension. The solution was boiled at 100℃ for
3 min. Next, 2.5 ml of the boiled cell suspension was
mixed with 2.5 ml of 4× cocktail primers (0.8 mM)
and 5 ml of 2× SYBR Premix EX Taq polymerase(Takara Bio). All cocktail primers were denatured
at 97℃ for 2 min. The following two-step PCR cycle
was applied 30 times for all primers: 60℃ for 10 s
and 97℃ for 5 s.
All JNBP strains of genus Shigella listed in Table
2 were analyzed for invE, stx1 (Shiga 1) and stx2
(Shiga 2). Reference serotyping strains, including 13
Shigella dysenteriae, 28 Shigella flexneri, 26 Shigellaboydii and 10 Shigella sonnei, were analyzed. dnaJ
primer to detect both Shigella species and E. coli
was also included in the cocktail primers.
Salmonella enterica serovar Typhi and serovar
Paratyphi A are SPJID pathogens. The invA geneis shared among all 2000 Salmonella serovars. To
differentiate these two serovars from other
Salmonella serovars, Vi antigen regulator (vipR) andflagellar antigen gene (fliC:a) were prepared. Vi
antigen is specific for serovar Typhi, but it is often
deleted from the chromosome (Hashimoto et al.,
1996, Hashimoto & Khan, 1997). We therefore selected another differential factor, CRISPR2, which is a
recently reported serovar Typhi-specific factor thatwas found by complete genome analysis (Fabre etal., 2014). CRISPR1 and FliC:a were selected for differentiating serovar Paratyphi A. FliC:a is a stablegene, but several other salmonella serovars belonging to group A flagellar groups cross-react with
FliC:a primers. CRISPR-1 has thus far been detected only in serovar Paratyphi A (Fabre et al., 2014).
CT toxin of
A cholera toxin (CT) and DnaJ chaperone gene

─ 124 ─Protein(gene)
Positive species

Forward─ 125 ─Hayashi, 2013
Hayashi, 2013

Tag_2-TCTGTATTTGCCGAAAACGT
Tag_1-GCCAGTTATCTGACATTCTG

This study
Fabre, 2014

Tag_2-CAGGGCATTTAACAGGCTGTAGCGA
Tag_1-GCGTGTAGCAGTATTCACCA

Tag_3 Phagetransposase
Burkholderia
Tag_1 Phage
mallei group
integraseActin
Tag_7 Actin

Yersinia pestis
group
Tag_3 F1 (caf1)
Tag_1 Pla (pla)

Tag_4 DnaJ (dnaJ)

Tag_1 Noncoding1

Tag_7-ATGGTGATGACCTGGCCGInternal control

PolyA-CGGGAAATCGTGCGTGAC

Janse, 2013

Tag_1-GCGTTCCACGATCAACTCThis study

This study
This study
Janse, 2013

Tag_3-TGGGATCACCCGCGGCATCTG
Tag_1-ACCTCCTTTCCACAGACATCC
Tag_3-AGCGGGTAGTCGAAGCTGCA

This study

Y. pestis, Y. pseudotu- PolyA-GATCCGAATCCCAACGCTGG
berculosis
Yersinia pestis
PolyA-GTTGGTACGCTTACTCTTGGCG
Yersinia pestis
PolyA-ATACTGTGACGGCGGGTCTGCA
B. mallei, B. pseudom- PolyA-CCGATTGGCGCTTCGTGCGC
allei
PolyA-GTGGGCGAAAGAACGCGAAC
B. mallei

Tag_4-TGGGCAAGTGACTGGTGAACTCThis study

Gunnell, 2012
Tag_4-AAAGCTGGCGATCCAAGG
F. tularensis subsp. PolyA-AGCTTATGCATCGAGTTGAGGTA
tularensis I
F. tularensis subsp. PolyA-CGAGATTTTGTCCACGCTTC
Tag_3-TTTGCGCCAAGACCAGAG
Gunnell, 2012
tularensis II
Gunnell, 2012
F. tularensis subsp. PolyA-TTGCCTATCCAATACTCCGAGTTAG Tag_2-CAAGCGCCTGGCTTTGATAA
holarctica
F. tularensis subsp. PolyA-TCAATGTTGCTAAAGTCTCTGGAG Tag_1-AATGATGGTAATAAAAGAAGTGGAGC Gunnell, 2012novicida

Tag_4 Noncoding4

This study

Fabre, 2014

Tag_3-GCATCATCGCGCATAGTGTC

Tag_5-GAAGAAGCGGTTCGCGGCTGC
Tag_1-TCCCGTCTGAGTTCCTCTTGC

This study

Tag_5-AGATAAGACGGCTGGTACTGA
Tag_4-CGCATCATCAAGGCCTGAGGCA

V. cholerae
V. cholerae O1, O139
PolyA-ACCTGATCCATCGCAAGCGTCA
PolyA-GGTCTTATGCCAAGAGGACAGAG

Hayashi, 2013

Tag_4-GCCAGTTATCTGACATTCTG

Hayashi, 2013This study

SourceTag_5-AGATAAGACGGCTGGTACTGA

Reverse

Tag_5 DnaJ (dnaJ)
Tag_1 CT (ctxA)

Tag_3 Noncoding3
Francisella tularensis group
Tag_2 Noncoding2

Vibrio cholerae

Salmonella spp
PolyA-TGACAGAATCCTCAGTTTTTCA
S. enterica serovar PolyA-GAACGTGCAGAAAGCGTATGA
Paratyphi A
S a l m o n e l l a Tag_3 CRISPR1
S. enterica serovar PolyA-ACGGGTTGCTGTAATGATGC
enterica serovar
Paratyphi A
Typhi group
Tag_2 VipR (vipR） S. enterica serovar PolyA-TCGGGGTTGGAGCTGCTGGCATTA
Typhi
Tag_1 CRISPR2
S. enterica serovar PolyA-ACGTAGACTCATCCTCGACC
Typhi

Tag_5 InvA (invA)
Tag_4 FliC

Tag_5 DnaJ (dnaJ)

Tag
position

E. coli, E. albertii, E. PolyA-TGACAGAATCCTCAGTTTTTCA
fegusonii, Shigella spp.
Tag_4 InvE (virB) E. coli EIEC, Shigella PolyA-GATACAGAGAGAATTTCGTC
Escherichiaspp.
Shigella group
PolyA-GATACAGAGAGAATTTCGTC
Tag_2 Shiga2 (stx2) E. coli EHEC
Tag_1 Shiga1 (stx1) E. coli EHEC, S. dys- PolyA-ACAGGATTTGTTAACAGGAC
enteriae O1Species

Table 1 Pathogenic factors and cocktail primers

Microb. Resour. Syst. Dec. 2016
Vol. 32, No. 2

Cocktail PCR and DNA strip for quick confirmation of pathogenic factors

cocktail was prepared to detect both CT-positiveand -negative strains of V. cholerae. Strains belonging to the O1 and O139 serotypes are generally bothctx- and dnaJ-positive. The other 140 serotypes
were expected to be CT-negative and dnaJ-positive.

Anti-tag 7 (I.C.)
Anti-tag 4
Anti-tag 3

Anti-tag 5
Anti-tag 2
Anti-tag 1

Ezaki et al.

andofBoth the plasmid-encoded plasminogen activatorgene pla (Guinet & Carniel, 2012) and the chromosomal chaperone gene dnaJ of Y. pestis were prepared. pla was recently reported in a wild strain ofEscherichia coli and in Citrobacter koseri (Hänsch etal., 2015). Pathogenic wild-type strains of Y. pestis
were expected to be both pla- and dnaJ-positive, but
plasmid-cured strains were expected to be only
dnaJ-positive. Because another plasmid-encoded
specific F1 antigen gene, caf1 (Galyov et al., 1990;
Tsui et al., 2015), produced nonspecific amplicons,
calf1 was not mixed with pla, but was instead amplified independently from the pla and dnaJ cocktail.
After PCR, the two amplicons were combined anddetected with a single DNA strip. The closely related Y. pseudotuberculosis has an identical dnaJ
sequence, and the dnaJ PCR was expected to bepositive. Once the plasmids are cured from Y. pestis, it cannot be differentiated from Y. pseudotuberculosis by using this cocktail primer (Table 3).
Subspecies and pathovar differentiation ofFig. 1 A DNA strip for differentiating six cocktail
PCR amplicons. Six anti-tags and three marker
lines are printed on a nitrocellulose membrane todifferentiate six PCR amplicons. Anti-Tag 7 isinternal control (I.C.)

Genome analyses have revealed sequences for differentiating subspecies of Francisella tularensis(Gunnell et al., 2012). We added tags to these four
primers and made a cocktail to differentiate two
subspecies, namely F. tularensis subsp. holarctica
and F. tularensis subsp. novicida, from F. tularensis

Table 2 Pathogenic factors and percentage of positive results
SpeciesVirulent factor% of positive results
among stock strains

Shigella dysenteriae
Shigella dysenteriae
Shigella flexneri
Shigella boydii
Shigella sonnei
Salmonella enterica serovar Typhi
Salmonella enterica serovar Typhi
Salmonella enterica serovar Paratyphi
Salmonella enterica serovar Typhi
Salmonella enterica serovar Paratyphi
Vibrio cholerae O1/O139
Vibrio cholerae other serotypes
Yersinia pestis
Yersinia pestis
Yersinia pseudotuberculosis

stx1
InvE
InvE
InvE
InvE
vipR
CRISPR2*CRISPR2*CRISPR1*CRISPR1*
ctxa
ctxa
caf1
pla
pla

8% (1/13)
15% (2/13)
25% (7/28)
31% (8/26)
20% (2/10)
85% (17/20）
100% (3/3)
0% (0/3)
0% (0/5)
100% (3/3)
87% (13/15)
0% (0/140)
60% (3/5)
60% (3/5)
0% (0/5)*CRISPAR1, CRISPAR2, are not pathogenic factors but cited in this table.
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Microb. Resour. Syst. Dec. 2016

Vol. 32, No. 2

Table 3 Differentiation of closely related subspecies and serovars
Francisella tularensis groupsubsp. tularensis GTC 3P0421
subsp. holarctica GTC 3P0422
subsp. novicida GTC 3P0425T
Salmonella enterica subsp. enterica group
serovar Typhi GTC 3P0637 Vi positive
serovar Typhi GTC 3P0612 Vi negative
serovar Paratyphi A GTC 3P0433
serovar Dublin GTC 02558 Vi positive
Citrobacter freundii GTC 14916 Vi positive
Bukholderia mallei groupB. pseudomallei GTC 3P0028
B. pseudomallei 10 wild-type strains
B. mallei GTC 3P0003T
B. mallei 4 wild-type strains
B. thailandensis GTC 3P0407T
B. vietnamiensis GTC 3P0436T
Yersinia pestis groupP. pestis GTC 3P0417T
P. pestis Russian vaccine strain
P. pestis 3 Mongolian wild-type strains
P. pseudotuberculosis GTC 03445

Tag 1
Noncoding1

Tag 2
Noncoding2

Tag 3
Noncoding3

Tag 4
Noncoding4

Tag 5
dnaJ

−
−
＋

−
＋
−

−
−
−

＋
−
−

＋
＋
＋

Tag 1

Tag 2

Tag 3

Tag 4

Tag 5

CRISPR2vipR

CRISPR1

FliC:a

invA

＋
＋
−
−
−

＋
−
−
−
−

−
−
＋
−
−

−
−
＋
−
−

＋
−
＋
＋
−

Tag 1

Tag 2

Tag 3

Tag 4

Tag 5

Tag 5

Phage
integrasePhagetransposase−(0/10)
+
(4/4)
−
−

＋
(10/10)
＋
(4/4)
−
−

Tag 1

Tag 3

Tag 4

pla

caf1

dnaJ

＋
＋
(2/3)
−

−
not tested
not tested
−

＋
＋
(3/3)
＋

subsp. tularensis.
Differentiation and identification ofandBurkholderia mallei and Burkholderia pseudomallei are genetically similar organisms (Yabuuchi et al.,1992). Housekeeping genes that are useful for differentiating two such closely related species are rare.
Several multiplex PCR methods have been reported(Rattanathongkom et al., 1997; Wongratanacheewin
et al., 2000), but closely related species, such as
Burkholderia thailandensis and environmental isolates of B. pseudomallei are not clearly differentiable. However, a recent whole-genome analysis hasrevealed several sequences that are specific to eachspecies (Janse, 2013). Using information on thesenoncoding sequences, we prepared tagged primers
to make cocktail PCR mixtures to differentiate thetwo species. Phage transposase, which is common
to both species, and phage integrase, which is foundTag 2

only in B. mallei (Table 1), were prepared for differentiating the two closely related species.

RESULTS AND DISCUSSION

The standard PCR method employs a high-profile
PCR tube to run 20 to 50 ml of PCR reaction mixtures, and 96 specimens can usually be amplified
with a standard PCR machine.
To reduce the amplification time, we designed afour-well PCR machine (Quick Mobile), andemployed low-profile PCR tubes to run experimentswith a 10 ml reaction volume. The DNA extraction
protocol was also simplified. Bacteria from a single
colony were suspended in 500 ml of distilled water(to a concentration of around 107 to 108 cfu/ml) andboiled for 3 min. With this simple sample preparation, the entire experiment could be completed within 40 min from the time bacteria were collected froma single colony. Gram-positive Mycobacterium tuberculosis and Clostridium botulinum, which have

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Cocktail PCR and DNA strip for quick confirmation of pathogenic factors

Ezaki et al.

and ethidium bromide staining; the amplicon could
be detected at dilutions up to eight-fold (Data notshown.)The PCR product from cell suspensions of S.
enterica serovar Typhi GTC 3P0637 at 108 to 103 cfu/
ml could be clearly visualized by using the DNAstrip method (Fig. 3A). However, the real-time PCR
results for the solutions at 104 to 102 cfu/ml could
not be differentiated from that for the negative con-tough cell walls, were successfully analyzed byusing this simple protocol, (Data not shown).
Sensitivity of the DNA strip
The PCR amplicon of the E. coli Shiga2-positive
strain O157:H7 (GTC 14509) was diluted by two-foldserial dilution. Up to 64-fold diluted amplicon was
visualized (Fig. 2). PCR amplicon in the same diluted solutions was also detected by electrophoresis

I.C.
Stx2

0

2

4

8

16

32

64

N.C.

Fig. 2 Sensitivity of the DNA strip
Serially diluted PCR amplicons of Escherichia coli Shiga2positive strain O157:H7 (GTC 14509) were detected with a standard latex solution. The DNA-strip method could detect theamplicon in all dilutions up to the 64-fold dilution.

I.C.

A

invA

108

107

106

105

104

103

102 101 cfu/ml

108, 107, 106,105 cfu/ml
B
104, 103 , 102, 101 cfu/ml
Negative control

Fig. 3 Real-time PCR and cocktail PCR DNA strips
A. T he PCR products from the cell suspensions of Salmonella enterica GTC
3P0637 at 108 cfu/ml to 103 cfu/ml could be clearly visualized using the DNAstrip method.
B. R eal-time PCR using the cell suspensions at 108 cfu/ml to 105 cfu/ml could
detect amplicon, but the method could not differentiate the cell suspensions at
104 cfu/ml to 102 cfu/ml. The Tm of the invA amplicon is 85.1℃.
─ 128 ─

Microb. Resour. Syst. Dec. 2016

Vol. 32, No. 2trol (Fig. 3B).
Screening for pathogenic factors
Freshly isolated Shigella species usually carry the
invE (virB) plasmid. Among 76 stock strains ofFig. 4 Cocktail to amplifyandFreshly isolated Shigella species usually carry the invE
plasmid. Among the 76 stock strains of Shigella species
analyzed, only 25% of the strains were invE-positive.
Shigella dysenteriae O1 GTC 00786 was the only strain
that was stx1-positive. Data for S. dysenteriae O1 GTC
00786 and the following representative stock strains areshown.
1. Escherichia coli (O157:H-) GTC 14508 (Shiga1+Shiga2+dnaJ),
2. S higella dysenteriae (O1) GTC 00786 (Shiga1+invE+
dnaJ）
3. Shigella flexneri GTC 01924 (invE+dnaJ)
4. Shigella flexneri GTC 01927 (invE negative+dnaJ)
5. Shigella boydii GTC 14835 (invE negative+dnaJ)

Shigella species, only 25% were invE-positive, andonly the Shigella dysenteriae O1 GTC 00786 strain
was stx1-positive (Table 1). Data for S. dysenteriae
O1 GTC 00786 and other representative stock
strains are shown in Figure 4.
The Vi antigen is generally believed to be specificto S. enterica serovar Typhi. However, some strainsof S. enterica serovar Dublin and Citrobacter freundii also carry Vi genes. The vipR primer that we
prepared did not amplify the DNA from Vi genepositive S. enterica serovar Dublin GTC 02558 or C.
freundii GTC 14916 (Fig. 5). Salmonella entericas e r o v a r T y p h i G T C 10007-1 w a s o r i g i n a l l y
Vi-positive, but the strain has lost its Vi gene (GTC
10007-3) (Hashimoto et al., 1996). Among the 20
stock strains analyzed, three had lost the Vi antigen(Table 2). The recently described Crispr-2 andCrispr-1 were specific to each serovar (Table 3).
The CT toxin of V. cholerae is reportedly present
only in serotypes O1 and 139 (Shi et al., 1998). The
CT gene is reported to be stable, but two strains
among 15 stock cultures of O1 and O139 wereCT-negative (Table 2). The non O1-O139 V. cholerae were all CT-negative among the 140 strains analyzed (Table 2).
The Fl antigen (caf1) and pla genes of Y. pestis
are located on plasmids, and these genes are often
cured from the host. The type strain GTC 3P0417
of Y. pestis had already lost the caf1. Among fourFig. 5 Cocktail to differentiate closely related
serovars and1. S. enterica serovar Typhi GTC 10007-1 (Vi+ invA)
2. S. enterica serovar Typhi GTC 10007-3 (del Vi, invA)
3. S. enterica serovar Dublin GTC 02558 (Vi positive)
4. C. freundii GTC 14916 (Vi positive)
5. S. enterica serovar Paratyphi A GTC 3P0433
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Cocktail PCR and DNA strip for quick confirmation of pathogenic factors

strains isolated from animals in Mongolia, threewere pla-positive, but one strain had lost pla (Table3). The dnaJ primer amplified sequences in both Y.
pestis and the closely related Y. pseudotuberculosis.
Both F1 antigen-gene and pla-deleted strains werepositive for dnaJ, and it was difficult to differentiate
Y. pestis from Y. pseudotuberculosis by using PCRbased methods.
The three subspecies of F. tularensis need to bedifferentiated from each other, because only F. tularensis subspecies tularensis and subspecies holarctica
are classified as SPJID pathogens. Housekeeping
genes such as the 16S rRNA gene and dnaJ geneare almost identical among species (Siddaramappa etal., 2012). Sequences specific to subspecies havebeen found by complete genome analysis (Gunnell etal., 2012). Four cocktail primers designed from thesegenomic sequences could be used to differentiate
each of the three subspecies and the wild-typestrains (Table 3).
Burkholderia pseudomallei and B. mallei are
genetically similar species (Yabuuchi et al., 1992) andthe 16S rRNA gene and housekeeping genes cannot
be used to differentiate them. Burkholderia pseudomallei is a motile species, whereas B. mallei is highly adapted to its host animals and has lost its motility. Complete genome analysis, has revealed many
phage-derived genes in their chromosomes (Janse etal., 2013). Phage-derived sequences and insertion
sequences were selected to differentiate the two
species. Primers for amplifying the phage integrase
in B. mallei did not amplify any sequences in B.
pseudomallei (Table 2). However, primers for the
integrase in B. pseudomallei could amplify sequences in all clinical strains. These two integrase primer
sets did not amplify any sequences in the closely
related B. thailandensis.

CONCLUSIONSThe cocktail PCR and DNA-strip method for differentiating six different amplicons is simple to perform. The same device can be used to detect pathogenic factors in BLS3 and SPJID pathogens. Culturecollection involves the handling of a broad range ofdifferent pathogens. Such collections are generally
maintained by only a few staff members, who are
expected to offer pathogenic information on all items
in collections. This quick and simple method of
detecting pathogenic factors in BLS3 and SPJID
strains in less than an hour is expected to be very

Ezaki et al.

useful in the handling of culture collections.

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BSL ３の保存細菌株の病原因子を迅速に確認するカクテル PCR-DNA クロマト法
江崎孝行1），金澤

泉1），林佐代子3），林1）将大2），エルデソキー・イブラヒム1），福永

肇1）

岐阜大学大学院医学研究科再生分子統御学講座，

岐阜大学生命科学センター嫌気性菌実験施設，3）岐阜医療科学大学

2）

BSL3 の細菌の分譲にあたって分譲株が不安定な病原性因子を保有しているかどうかの情報が要求される．多種類にわたる菌種の病原因子を確認し，分譲することは少数のスタッフで運営されている分譲業務には大きな負担になる．我々は最大6 種類のプライマーに Tag をつけ，一本の PCR チューブ内で増幅するカクテルプライマー増幅法を作成し，増幅産物をDNA-Strip 上にプリントした Anti-Tag に結合させ，3 本の位置マーカー情報と対比させ，増幅産物を目視判定する方法を作成した．この方法で単一集落から PCR 産物の目視判定まで 40 分以内で BSL3 の病原体の病原因子の有無を判定する方法を確立した．

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