Curr Microbiol (2014) 68:486–494 DOI 10.1007/s00284-013-0488-1
A Simple One-Step PCR Walking Method and Its Application of Bacterial rRNA for Sequencing Identification Hongfa Zhang • Chunping You • Jing Ren Dan Xu • Mei Han • Wenyan Liao
•
Received: 4 January 2013 / Accepted: 28 September 2013 / Published online: 21 November 2013 Ó Springer Science+Business Media New York 2013
Abstract There are many PCR walking methods applied currently, and they all have examples of successful application in organisms which are more complex than bacteria. However, to a certain extent, it will be more convenient for researchers if the complicated operation and poor specificity for bacteria can be improved. Here, we introduced an improved one-step PCR walking method of bacteria. Using a specific primer of the known sequence together with a universal semirandom primer, the unknown sequence adjacent to a known sequence can be obtained easily by just one ordinary round PCR. The products can be gel purified and directly sequenced. Specific primers were designed according to the gene sequence of bacterial rRNA, and the variable and adjacent gene sequences were obtained by this method. The sequence analysis of the product showed that it can improve the resolution of bacterial identification to the species level.
Introduction It is an important task in microbial genome-related research to obtain unknown sequence adjacent to a known sequence using gene walking [6]. There are many PCR-based gene
H. Zhang (&) C. You J. Ren M. Han W. Liao State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy & Food Co., Ltd, Shanghai 200436, China e-mail: [email protected] D. Xu Pass College of Chongqing Technology and Business University, Chongqing 400060, China
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walking methods. Although there are examples of successful application of these methods, most of them are complicated in operation. For example, self-formed adaptor PCR is efficient to get unknown sequence in complex genome, but the cycling conditions are complicated, and the annealing temperatures of several cycles are ranged from 35 to 45 °C [12]; sequential hybrid primer PCR has two rounds of PCR, and the annealing temperature of one of the cycles is 35 °C [8]; two-step PCR has 61 cycles, and the annealing temperature for one of the cycles is 40 °C [10]; easy gene walking has to use three rounds of PCR to get the target sequence, although the annealing temperature is higher [3]. In short, to some extent, almost all the gene walking methods currently used have inconveniences of complicated operation and poor specificity for bacteria which genome is relatively small. Because the lower annealing temperature likely to increasing nonspecific binding of the primer with the template, and increasing the nonspecific products of amplification. Therefore, it is urgent that we need to explore a simple, efficient, and universal gene walking method for bacterial identification. Sequences of rRNA genes have been well-applied in microbial identification [9]. However, since their sequences are relatively conserved [5, 14], it is almost impossible to discriminate between highly related species [2]. Also, as rRNA genes in bacteria often occur in multiple copies [4], PCR products of rRNA genes are often mixed, and hence they have to be cloned and sequenced. In this study, a one-step PCR walking method was established, and primarily used to amplify variable regions of multiple copies gene which are adjacent to conserved regions of rRNA in bacteria. After being gel purified, they were directly sequenced and analyzed for bacteria identification.
H. Zhang et al.: A Simple One-Step PCR Walking Method
487
Table 1 Primers Primer name
Primer sequence(50 –30 )
About gene
357R
CTGCTGCCTCCCGTAGGAG
16S
474F
AGTACCGTGAGGGAAAGG
23S
474R
CCTTTCCCTCACGGTACT
23S
DQW
GTCGGCGTTTATTCAGAAG
AP
GTCGGCGTTTATTCAGAAG(N)6GACGCC
EU27F
AGAGTTTGATCM(C/A)TGGCTCAG
16S
1492R
TACGGY(C/T)TACCTTGTTACGACTT
16S
Materials and Methods Primers Primers used in this study are shown in Table 1. The primers of rRNA were designed for members of bacteria, but not for those of archaea. EU27F and 1492R were designed according to references of [1, 7], 357R was designed according to references of [13, 14], 474F and 474R were designed according to reference of [2]. The semirandom primer (AP) consists of three parts (50 –30 ): (i) DQW primer, (ii) 6 bases of degenerate sequence, and (iii) 6 bases of a specific sequence. DQW was the sequence of the nonbacterial strains. The specificity of the primers was checked using BLAST in NCBI. One-Step PCR Walking The principle of one-step PCR walking method is shown in Fig. 1 [10, 11]. Firstly, specific primers (SP) only amplify the single chain of the target gene sequence at the higher annealing temperature. When single chain accumulates to a certain number, they combine with the AP, and are amplified with SP and AP. The nonspecific pairing of AP and SP is reduced at higher annealing temperature, and that reduce nonspecific amplifications. SP or AP which containing the sequence of DQW can
form panhandle structure to inhibit the nonspecific amplification. Genomic DNA of single colonies of bacterial strains was isolated from bacteria using TaKaRa minibest bacterial genomic DNA extraction kit ver. 2. 0 (Takara Biotechnology (Dalian) Co., Ltd). Approximately, 50 ng of template DNA was used for PCR amplification with SP (0.5 lmol/L) and AP (0.5 lmol/L) in a 50 lL reaction volume, 5u Ex-Taq (Takara Biotechnology (Dalian) Co., Ltd). The cycling program was as follows: 94 °C for 5 min; 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; and extended reaction at 72 °C for 5 min. The clear bands of the remaining PCR products were gel purified and sequenced (Sagon, Shanghai, China) directly by using the primer of either SP or DQW. The results were analyzed by BLAST in NCBI.
Results Selection of the Primers of rRNA for Bacterial Identification Genomic DNA of 23 strains bacteria of 18 species was used as templates for PCR walking to get sequences about rRNA. 357R primer was used to get upstream sequence of 16S rRNA, 474R primer was used to get intergenic sequence of 16S-23S rRNA, and 474F primer was used to get sequence of 23S rRNA. The results (Fig. 2; Table 2) prove that one-step PCR walking method can be used for different primers and bacteria. The sequences obtained from primer 357R of the 23 bacteria show that only one species of bacteria was the highest homologous, and can be used for bacterial identification. Identification of Different Bacteria The primer 357R was used to obtain upstream sequence of 16S rRNA. Using the primers of EU27F and 1492R, to obtain the sequence of 16S rRNA. The results of
Fig. 1 Basic principle of the AP gene-walking PCR. The known sequence stretch is shown as solid line, the unknown sequence stretch is shown as dotted line, SP is shown as black arrow, and AP is shown as black and white arrow. After 35 cycles, the target sequences were the products of the double primers of SP and AP; the nontarget sequences were the products of a single prime of SP or AP
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H. Zhang et al.: A Simple One-Step PCR Walking Method
Fig. 2 Walking PCRs of different bacterial rRNA. Lane M: DL2, 000 DNA Marker; Lanes 1–3:357R, 474F, 474R. The bands shown by arrow were purified and sequenced
BLAST are shown in Table 3. For the BLAST analysis of the sequence upstream 16S rRNA, the bacterium with the highest score is corresponding to the bacterium of detection, and is significantly different from the other bacteria with the second highest score. All 41 selected strains of 31 species can be identified at the species level.
Discussion Compared with the ordinary walking PCR methods, the one-step PCR walking in this study has the advantages of convenient operation. Furthermore, the higher annealing temperature in the entire PCR procedure ensures the specificity and accuracy of the amplification. This is due to the fact that the AP primer consists of three parts (50 –30 ): 1.
The DQW primer, which was used as sequencing primer, can increase the pairing length of the APs to amplify the target sequence in the first round; DQW also can help forming panhandle structure
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between primers of AP to inhibit the nonspecific amplification. 2. Six bases of degenerate sequence add more complementary sites to increasing annealing temperature. 3. Six bases of a specific sequence have high G/C % which can increase annealing temperature. As sequences of rRNA genes are obtained the universality of bacterial sequencing primers, and the variability of amplified sequences is achieved. It can also solve the problem that the mixed PCR products of multiple gene copies cannot be directly sequenced. The resolution of bacterial identification by using the primer 357R was increased to the species level. In practical applications, we also found that some bacteria sequences do not contain the corresponding upstream sequences of the 16S rRNA genes deposited in public databases. This leads to difficulties in identifying highly related sequences by BLAST analysis as often several sequences show the highest BLAST scores. With the speed at which genomes of bacterial strains are sequences it will one day be unnecessary to do PCR at all as relationships will be determined by IT programs.
Streptococcus thermophilus St-body 3
Streptococcus thermophilus TA40(St01)
Lactobacillus delbrueckii ssp.bulgaricus LB-12
Lactobacillus delbrueckii ssp.bulgaricus LB340(LB340)
Lactobacillus casei 01(LC01)
Lactobacillus casei Shirota(LCYA)
Lactobacillus casei LC2W(LC2W)
357R
Lactobacillus casei 431(LC431)
DQW-5
Sequencing primer
DQW-5 DQW-5
474R 357R
100 474R
474R
KC161281 (768)
99.9
100 KC161271 (748)
474F
100
474F
KC161292 (624)
KC161280 (548)
The first 357 nt of 16S rRNA (301 bp)
357R
474R
474R 357R
100 99.3
100 KC161270 (283)
474F
99.9
99.6
The first 357 nt of 16S rRNA (285 bp)
KC161291 (717)
KC161279 (811)
474F
357R
474R
474R 357R
100 99.9
474F
KC161269 (756)
The first 357 nt of 16S rRNA (83 bp) 474F
KC161278 (275) KC161297 (605)
97.5 99.2
99.3
KC161267 (452)
474F
474F
98.5
100 100
KC161296 (600)
KC161277 (735)
The first 357 nt of 16S rRNA (74 bp)
357R
DQW-5
474R 357R
100
KC161266 (713)
DQW-5
100
474F
KC161261 (695) No
DQW-5
The first 357 nt of 16S rRNA
357R
474R
474R
100
100
KC161276 (856)
No KC161265 (688)
99.7
474F
KC161260 (664)
99.3
100
100 No
99.9
100
No
95.5
Identities of the target bacteria (%)
474F
DQW-5
KC161275 (544)
KC161264 (718)
KC161289 (578)
KC161274 (786 )
KC161263 (715)
KC161259 (779)
Accession number (length) (bp)
The first 357 nt of 16S rRNA
357R
DQW-5 DQW-5
474F 474R
357R DQW-5 The first 357 nt of 16S rRNA
DQW-5 DQW-5
474F 474R
The first 357 nt of 16S rRNA
Walking primer
Bacterial name
Table 2 Selection of the primers about rRNA for bacterial identification
99.2 (Streptococcus salivarius)
99.9 (Streptococcus salivarius)
99.7 (Streptococcus salivarius)
94.2 (Streptococcus salivarius)
98.5 (Streptococcus salivarius)
100 (Streptococcus salivarius)
99.6 (Streptococcus salivarius)
98.9 (Streptococcus salivarius)
83.5 (Lactobacillus helveticus)
91.7 (Lactobacillus helveticus)
91.6 (Lactobacillus helveticus)
75.3 (Lactobacillus helveticus) 13.1 (Lactobacillus kefiranofaciens)
92.5 (Lactobacillus helveticus)
100 (Lactobacillus acidophilus)
13.7 (Lactobacillus kefiranofaciens)
98 (Lactobacillus rhamnosus)
98.6 (Lactobacillus rhamnosus)
No
82.3 (Lactobacillus rhamnosus)
97.9 (Lactobacillus rhamnosus)
98.5 (Lactobacillus rhamnosus)
No
80.7 (Lactobacillus rhamnosus)
96.9 (Lactobacillus rhamnosus)
98.6 (Lactobacillus rhamnosus)
94.5 (Lactobacillus rhamnosus) No
97.8 (Lactobacillus rhamnosus)
96.8 (Lactobacillus rhamnosus)
No
84.5 (Lactobacillus rhamnosus)
Identities of the nontarget bacteria (%)
H. Zhang et al.: A Simple One-Step PCR Walking Method 489
123
123
Corynebacterium glutamicum ACCC16522
Bifidobacterium longum subsp. longum CICC 21717
Bacillus pumilus CMCC63202
Bacillus megaterium ACCC11011
Bacillus cereus ATCCC1220
Lactobacillus.acidophilus NCFM(NCFM)
Escherichia coli DH5a
KC161294 (685)
KC161282 (696)
357R
357R
KC768812 (381)
99.0
100
357R
KC768811 (375)
KC768839 (630)
474F 474R
474F 474R
KC768840 (454)
\200 bp (131)
The first 357 nt of 16S rRNA(242 bp)
357R
474R
474R
100
100
99.6
94.7
99.8
100
KC768830 (659)
100
99.5
474F
KC415589 (804)
\200 bp (187)
The first 357 nt of 16S rRNA(283 bp)
357R
474R
474F
357R
474R
99.7 99.8
The first 357 nt of 16S rRNA(297 bp) 474F 474F KC768832 (420)
97.9
\200 bp (141)
98.1
100
\200 bp (150)
99.6
KC768816 (424)
357R
474R
474R 357R
474F
474F
The first 357 nt of 16S rRNA(245 bp)
99.6
100
KC768834 (568)
KC768827 (909)
474F 474R
474F
97.9
474R
KC415592 (426) 97.3
357R
KC161284 (671)
The first 357 nt of 16S rRNA(188 bp)
357R
DQW-5
474R
99.6
99.8
KC161273 (427)
100
100
100 99.8
474F
KC161295 (743)
\200 bp (127) KC161283 (573)
The first 357 nt of 16S rRNA(299 bp)
357R
474F 474R
100
100
100
474F
357R
474F 474R
The first 357 nt of 16S rRNA(311 bp)
DQW-5
474R
357R
474R
KC161272 (681)
99.9
100
474F
KC161293 (748)
Identities of the target bacteria (%)
474F
357R
Accession number (length) (bp)
100
357R
Lactobacillus Plantarum ST-III(ST-III)
Sequencing primer
The first 357 nt of 16S rRNA (318)
Walking primer
Bacterial name
Table 2 continued
No
29.0 (Corynebacterium urealyticum)
85.1 (Rhodococcus globerulus)
54.9 (Rhodococcus globerulus)
99.7 (Bifidobacterium longum subsp. infantis)
100 (Bifidobacterium longum subsp. infantis)
100 (Bifidobacterium longum subsp. Infantis)
75.6 (Bifidobacterium breve)
93.6 (Bacillus atrophaeus)
97.3 (Bacillus safensis) 99.5 (Bacillus subtilis subsp)
68.1 (Bacillus safensis)
55.3 (Bacillus cereus subsp. cytotoxis)
95.3 (Bacillus thuringiensis serovar finitimus)
98.0 (Bacillus aryabhattai)
63.0 (Bacillus aryabhattai)
99.6 (Bacillus thuringiensis)
100 (Bacillus thuringiensis)
97.3 (Bacillus weihenstephanensis)
93.7 (Bacillus thuringiensis serovar finitimus)
97.2 (Lactobacillus amylovorus)
98.1 (Lactobacillus crispatus)
97.0 (Lactobacillus helveticus)
91.5 (Lactobacillus amylovorus)
100 (Shigella sonnei) 98.8 (Shigella sonnei)
99.7 (Shigella dysenteriae)
98.4 (Shigella flexneri)
92.5 (Lactobacillus pentosus)
99.3 (Lactobacillus pentosus)
100 (Lactobacillus pentosus)
49.3 (Lactobacillus pentosus)
Identities of the nontarget bacteria (%)
490 H. Zhang et al.: A Simple One-Step PCR Walking Method
Shigella sonnei CMCC51334
Shigella flexneri CCAM 090021
Salmonella Typhimurium ATCC14023
Lysinibacillus sphaericus ACCC11096
Lactococcus lactis subsp. lactis Il1403
Enterobacter cloacae ATCC13047
357R
KC768814 (329)
KC768838 (848)
357R
KC768831 (452) KC768817 (712)
KC768833 (415)
474R
100
KC768819 (705)
KC768835 (441)
474F 474R
474F 474R
KC768843 (604)
\200 bp (132)
The first 357 nt of 16S rRNA (300 bp)
357R
474R
100
100
100
100
100
357R
474R
99.3
100
100
100
100
100
100 100
KC415591 (539)
KC768841 (602)
\200 bp (128)
100 100
The first 357 nt of 16S rRNA (303 bp) 474F 474F \200 bp (127)
357R
474R 357R
474F
474F
The first 357 nt of 16S rRNA (283 bp)
357R
474F 474R
474F 474R
The first 357 nt of 16S rRNA (295 bp)
357R
474R
474R
100
100
KC768829 (886)
100
100
100 97.6
100
474F
KC415597 (764)
\200 bp (132) KC768842 (586)
The first 357 nt of 16S rRNA (311 bp)
357R
474F 474R
99.5 94.0
474F
357R
474F 474R
The first 357 nt of 16S rRNA (292 bp)
KC768826 (699)
KC768836 (611)
474R 357R
474R 357R
100
KC768828 (672)
99.3
474F
KC415588 (731)
Identities of the target bacteria (%)
474F
DQW-5
Accession number (length) (bp)
No
357R
Enterobacter aerogenes ATCC13048
Sequencing primer
The first 357 nt of 16S rRNA
Walking primer
Bacterial name
Table 2 continued
99.5 (Escherichia coli)
100 (Shigella sonnei)
100 (Escherichia coli)
99.3 (Escherichia coli)
99.5 (Shigella boydii)
99.7 (Escherichia coli) 100 (Escherichia coli)
98.1 (Escherichia coli)
99.5 (Salmonella typhimurium)
98.4 (Pectobacterium carotovorum subsp)
99.6 (Salmonella paratyphi)
95.5 (Salmonella bongori)
92.5 (Solibacillus silvestris)
97.3 (Solibacillus silvestris)
99.3 (Brevibacillus laterosporus)
89.1 (Brevibacillus laterosporus)
99.9 (Lactococcus lactis subsp. cremoris)
100 (Lactococcus lactis subsp. cremoris)
99.4 (Streptococcus lactis)
65.3 (Lactococcus lactis subsp. cremori)
100 (Pantoea endophytica) 96.2 (Klebsiella pneumoniae subsp. pneumoni)
100 (Escherichia coli)
93.0 (Escherichia coli)
90.3 (Enterococcus hirae)
98.4 (Enterococcus faecium)
No
83.0 (Klebsiella oxytoca)
Identities of the nontarget bacteria (%)
H. Zhang et al.: A Simple One-Step PCR Walking Method 491
123
123 100
KC768816 (424) KC768811 (375) KC768826 (699) KC161294 (685) KC768813 (576)
Corynebacterium glutamicum ACCC16522
Enterobacter cloacae ATCC13047
Escherichia coli DH5a
Kocuria rhizophila DC2201
KC161289 (578)
KC768812 (381)
Bacillus megaterium ACCC11011
Bacillus pumilus CMCC63202
KC161260 (664)
KC415589 (804)
Bifidobacterium longum subsp. longum CICC 21717
Lactobacillus casei LC2 W
KC415597 (764)
Lactococcus lactis subsp. lactis Il1403
Lactobacillus casei Shirota
100
KC415598 (719)
Lactobacillus casei ATCC 334
KC161261 (695)
KC415596 (699)
Lactobacillus casei str. Zhang
KC161259 (779)
KC415595 (764)
Lactobacillus plantarum WCFS1
Lactobacillus casei 01
KC415594 (761)
Streptococcus thermophilus LMD-9
Lactobacillus casei 431
100
KC415593 (700)
Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293
99.3
99.7
100
95.5
100
94.0
94.7
98.1
99.0
99.5
100
99.7
100
100
100
99.9
99.8
KC415588 (731) KC415599 (571)
Enterobacter aerogenes ATCC13048
99.3
Enterococcus faecalis ACCC10699
KC415590 (735) KC415591 (539)
Escherichia coli ATCC25922
Shigella flexneri CCAM 090021
99.2 97.9
KC415587 (766) KC415592 (426)
Bacillus licheniformis ACCC02002
Bacillus cereus ATCCC1220
99.0
KC415586 (491)
80.7 (Lactobacillus rhamnosus)
94.5 (Lactobacillus rhamnosus)
84.5 (Lactobacillus rhamnosus)
82.3 (Lactobacillus rhamnosus)
36.5 (Arthrobacter globiformis)
98.4 (Shigella flexneri)
93.1 (Escherichia coli)
54.9 (Rhodococcus globerulus)
68.2 (Bacillus safensis strain)
63.0 (Bacillus aryabhattai)
75.6 (Bifidobacterium breve)
65.3 (Lactococcus lactis subsp. cremori)
79.4 (Lactobacillus rhamnosus)
KC768796 (1,406)
KC768795 (1,399)
KC768794 (1,365)
KC768797 (1,310)
KC768807 (1,360)
KC768803 (1,356)
KC768786 (1,369)
KC768805 (1,355)
KC768810 (1,400)
KC768806 (1,402)
KC429787 (1,359)
KC429785 (1,411)
KC429784 (1,400)
KC429783 (1,425)
KC429782 (1,420)
85.4 (Lactobacillus rhamnosus)
KC429781 (1,381)
100 (Lactobacillus pentosus)a
KC429780 (1,402)
KC429779 (1,402)
KC429778 (1,376)
KC429777 (1,353)
KC429776 (1,355)
KC429775 (1,396)
KC429774 (1,393)
KC429773 (1,397)
80.7 (Streptococcus salivarius)
95.5 (Leuconostoc carnosum)
87.2 (Enterococcus hirae)
83.0 (Klebsiella oxytoca)
98.1 (Escherichia coli)
97.4 (Shigella flexneri)
93.7 (Bacillus thuringiensis serovar finitimus)
63.8 (Bacillus subtilis)
91.2 (Bacillus atrophaeus)
Accession number (length) (bp)
Identities of the nontarget bacteria (%)
Accession number (length) (bp)
Identities of the target bacteria (%)
Sequence of 16S rRNA
Sequence of 357R
Bacillus subtilis CMCC63501
Bacterial name
Table 3 BLAST analysis of different bacteria
100
100
100
99.9
100
100
100
100
99.9
100
100
100
100
100
100
100
100
100
100
100
100
100
99.0
100
Identities of the target bacteria (%)
100 (Lactobacillus paracasei)
100 (Lactobacillus paracasei)
100 (Lactobacillus paracasei)
99.9 (Lactobacillus paracasei)
99.8 (Arthrobacter globiformis)
100 (Shigella flexneri)
99.9 (Enterobacter asburiae)
99.4 (Corynebacterium acetoacidophilum)
99.9 (Bacillus safensis)
100 (Bacillus aryabhattai)
100 (Bifidobacterium longum subsp. Infantis)
100 (Lactobacillus paracasei;Lactobacillus rhamnosus) 99.9 (Lactococcus garvieae)
100 (Lactobacillus paracasei;Lactobacillus rhamnosus)
100 (Enterococcus durans)
99.8 (Streptococcus salivarius)
99.9 (Lactobacillus delbrueckii subsp. bulgaricus)
100 (Enterococcus faecium)
99.9 (Pantoea sp.)
100 (Escherichia coli)
99.9 (Shigella flexneri)
100 (Bacillus anthracis)
98.7 (Bacillus aerius)
100 (Bacillus tequilensis;Bacillus methylotrophicus)
Identities of the nontarget bacteria (%)
492 H. Zhang et al.: A Simple One-Step PCR Walking Method
KC768820 (711) KC768819 (705) KC768822 (445) KC161292 (624) KC161291 (717) KC768823 (342) KC768824 (805) KC768825 (644)
Shigella dysenteriae CMCC51335
Shigella sonnei CMCC51334
Staphylococcus aureus ATCC8095
Streptococcus thermophilus St-body 3
Streptococcus thermophilus TA40
Vibrio cholerae SJTU32001
Vibrio parahaemolyticus ATCC17802
Vibrio vulnficus ATCC27562
98.8
99.3
100
99.9
100
100
100
99.2
100
99.6
95.3
99.5
100
100
100
98.5
99.2
78.1 (Vibrio cholerae)
59.6 (Vibrio harveyi)
86.8 (Vibrio mimicus strain)
98.9 (Streptococcus salivarius)
94.2 (Streptococcus salivarius)
95.3 (Staphylococcus epidermidis)
99.3 (Escherichia coli)
98.3 (Escherichia coli)
95.5 (Salmonella bongori)
90.9 (Salmonella bongori)
93.4 (Pseudomonas brassicacearum subsp)
72.5 (Pseudomonas stutzeri)
89.1 (Brevibacillus laterosporus)
91.5 (Lactobacillus amylovorus)
49.3 (Lactobacillus pentosus)
13.7 (Lactobacillus kefiranofaciens)
13.1 (Lactobacillus kefiranofaciens)
KC768793 (1,361)
KC768792 (1,368)
KC768791 (1.363)
KC768800 (1,372)
KC768801 (1,374)
KC768790 (1,376)
KC768787 (1,362)
KC768788 (1,369)
KC768784 (1,367)
KC768785 (1,360)
KC768809 (1,386)
KC768789 (1,317)
KC768808 (1,377)
KC768804 (1,394)
KC768802 (1,400)
KC768798 (1,374)
KC768799 (1,381)
Lactobacillus pentosus MP-10 draft has no complete sequence in NCBI; the sequence of BLAST is too short (378/764)
KC768817 (712)
Salmonella Typhimurium ATCC14023
a
KC768818 (689)
Salmonella Heidelberg ATCC13311
KC768814 (329)
Lysinibacillus sphaericus ACCC11096 KC768821 (799)
KC161295 (743)
Lactobacillus.acidophilus NCFM
KC768815 (682)
KC161293 (748)
Lactobacillus Plantarum ST-III
Pseudomonas aeruginosa CDCB32116
KC161296 (600)
Lactobacillus delbrueckii ssp.bulgaricus LB340
Pseudomonas fluorescens AS1.823
KC161297 (605)
Accession number (length) (bp)
Identities of the nontarget bacteria (%)
Accession number (length) (bp)
Identities of the target bacteria (%)
Sequence of 16S rRNA
Sequence of 357R
Lactobacillus delbrueckii ssp.bulgaricus LB-12
Bacterial name
Table 3 continued
100
99.9
100
100
100
100
100
100
100
100
99.9
100
100
100
99.9
100
100
Identities of the target bacteria (%)
97.3 (Vibrio aestuarianus)
99.9 (Vibrio alginolyticus)
99.9 (Vibrio mimicus)
99.8 (Streptococcus salivarius)
99.8 (Streptococcus salivarius)
98.8 (Staphylococcus epidermidis)
99.9 (Escherichia coli)
99.7 (Escherichia coli)
99.1 (Salmonella paratyphi)
99.9 (Salmonella choleraesuis)
99.9 (Pseudomonas chlororaphis subsp)
99.9 (Rhodococcus erythropolis)
100 (Lysinibacillus fusiformis)
99.6 (Lactobacillus kitasatonis)
99.9 (Lactobacillus pentosus)
99.3 (Lactobacillus leichmannii)
99.7 (Lactobacillus delbrueckii subsp. sunkii)
Identities of the nontarget bacteria (%)
H. Zhang et al.: A Simple One-Step PCR Walking Method 493
123
494 Acknowledgments This work was supported by the Major Project of Chinese National Programs for Fundamental Research and Development (973 Program) (grant no. 2012CB723706).
References 1. Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ (2008) Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl Environ Microbiol 74(8):2461–2470. doi:10.1128/AEM.02272-07 2. Gurtler V, Stanisich VA (1996) New approaches to typing and identification of bacteria using the 16S–23S rDNA spacer region. Microbiology 142(Pt 1):3–16 3. Harrison RW, Miller JC, D’Souza MJ, Kampo G (1997) Easy gene walking. Biotechniques 22(4):650–653 4. Klappenbach JA, Saxman PR, Cole JR, Schmidt TM (2001) rrndb: the ribosomal RNA operon copy number database. Nucleic Acids Res 29(1):181–184 5. Ludwig W, Schleifer KH (1994) Bacterial phylogeny based on 16S and 23S rRNA sequence analysis. FEMS Microbiol Rev 15(2–3):155–173 6. Luo P, Su T, Hu C, Ren C (2011) A novel and simple PCR walking method for rapid acquisition of long DNA sequence flanking a known site in microbial genome. Mol Biotechnol 47(3):220–228. doi:10.1007/s12033-010-9332-z 7. Magray MS, Kumar A, Rawat AK, Srivastava S (2011) Identification of Escherichia coli through analysis of 16S rRNA and
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8.
9. 10.
11.
12.
13.
14.
16S–23S rRNA internal transcribed spacer region sequences. Bioinformation 6(10):370–371 Martin-Harris MH, Bartley PA, Morley AA (2010) Gene walking using sequential hybrid primer polymerase chain reaction. Anal Biochem 399(2):308–310. doi:10.1016/j.ab.2010.01.005 Olsen GJ, Woese CR (1993) Ribosomal RNA: a key to phylogeny. FASEB J 7(1):113–123 Pilhofer M, Bauer AP, Schrallhammer M, Richter L, Ludwig W, Schleifer KH, Petroni G (2007) Characterization of bacterial operons consisting of two tubulins and a kinesin-like gene by the novel two-step gene walking method. Nucleic Acids Res 35(20):e135. doi:10.1093/nar/gkm836 Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23(6):1087–1088 Wang S, He J, Cui Z, Li S (2007) Self-formed adaptor PCR: a simple and efficient method for chromosome walking. Appl Environ Microbiol 73(15):5048–5051. doi:10.1128/AEM.0297306 Wang Y, Qian PY (2009) Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PLoS One 4(10):e7401. doi:10.1371/journal.pone.0007401 Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173(2):697–703
A Simple One-Step PCR Walking Method and Its Application of Bacterial rRNA for Sequencing Identification Hongfa Zhang • Chunping You • Jing Ren Dan Xu • Mei Han • Wenyan Liao
•
Received: 4 January 2013 / Accepted: 28 September 2013 / Published online: 21 November 2013 Ó Springer Science+Business Media New York 2013
Abstract There are many PCR walking methods applied currently, and they all have examples of successful application in organisms which are more complex than bacteria. However, to a certain extent, it will be more convenient for researchers if the complicated operation and poor specificity for bacteria can be improved. Here, we introduced an improved one-step PCR walking method of bacteria. Using a specific primer of the known sequence together with a universal semirandom primer, the unknown sequence adjacent to a known sequence can be obtained easily by just one ordinary round PCR. The products can be gel purified and directly sequenced. Specific primers were designed according to the gene sequence of bacterial rRNA, and the variable and adjacent gene sequences were obtained by this method. The sequence analysis of the product showed that it can improve the resolution of bacterial identification to the species level.
Introduction It is an important task in microbial genome-related research to obtain unknown sequence adjacent to a known sequence using gene walking [6]. There are many PCR-based gene
H. Zhang (&) C. You J. Ren M. Han W. Liao State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy & Food Co., Ltd, Shanghai 200436, China e-mail: [email protected] D. Xu Pass College of Chongqing Technology and Business University, Chongqing 400060, China
123
walking methods. Although there are examples of successful application of these methods, most of them are complicated in operation. For example, self-formed adaptor PCR is efficient to get unknown sequence in complex genome, but the cycling conditions are complicated, and the annealing temperatures of several cycles are ranged from 35 to 45 °C [12]; sequential hybrid primer PCR has two rounds of PCR, and the annealing temperature of one of the cycles is 35 °C [8]; two-step PCR has 61 cycles, and the annealing temperature for one of the cycles is 40 °C [10]; easy gene walking has to use three rounds of PCR to get the target sequence, although the annealing temperature is higher [3]. In short, to some extent, almost all the gene walking methods currently used have inconveniences of complicated operation and poor specificity for bacteria which genome is relatively small. Because the lower annealing temperature likely to increasing nonspecific binding of the primer with the template, and increasing the nonspecific products of amplification. Therefore, it is urgent that we need to explore a simple, efficient, and universal gene walking method for bacterial identification. Sequences of rRNA genes have been well-applied in microbial identification [9]. However, since their sequences are relatively conserved [5, 14], it is almost impossible to discriminate between highly related species [2]. Also, as rRNA genes in bacteria often occur in multiple copies [4], PCR products of rRNA genes are often mixed, and hence they have to be cloned and sequenced. In this study, a one-step PCR walking method was established, and primarily used to amplify variable regions of multiple copies gene which are adjacent to conserved regions of rRNA in bacteria. After being gel purified, they were directly sequenced and analyzed for bacteria identification.
H. Zhang et al.: A Simple One-Step PCR Walking Method
487
Table 1 Primers Primer name
Primer sequence(50 –30 )
About gene
357R
CTGCTGCCTCCCGTAGGAG
16S
474F
AGTACCGTGAGGGAAAGG
23S
474R
CCTTTCCCTCACGGTACT
23S
DQW
GTCGGCGTTTATTCAGAAG
AP
GTCGGCGTTTATTCAGAAG(N)6GACGCC
EU27F
AGAGTTTGATCM(C/A)TGGCTCAG
16S
1492R
TACGGY(C/T)TACCTTGTTACGACTT
16S
Materials and Methods Primers Primers used in this study are shown in Table 1. The primers of rRNA were designed for members of bacteria, but not for those of archaea. EU27F and 1492R were designed according to references of [1, 7], 357R was designed according to references of [13, 14], 474F and 474R were designed according to reference of [2]. The semirandom primer (AP) consists of three parts (50 –30 ): (i) DQW primer, (ii) 6 bases of degenerate sequence, and (iii) 6 bases of a specific sequence. DQW was the sequence of the nonbacterial strains. The specificity of the primers was checked using BLAST in NCBI. One-Step PCR Walking The principle of one-step PCR walking method is shown in Fig. 1 [10, 11]. Firstly, specific primers (SP) only amplify the single chain of the target gene sequence at the higher annealing temperature. When single chain accumulates to a certain number, they combine with the AP, and are amplified with SP and AP. The nonspecific pairing of AP and SP is reduced at higher annealing temperature, and that reduce nonspecific amplifications. SP or AP which containing the sequence of DQW can
form panhandle structure to inhibit the nonspecific amplification. Genomic DNA of single colonies of bacterial strains was isolated from bacteria using TaKaRa minibest bacterial genomic DNA extraction kit ver. 2. 0 (Takara Biotechnology (Dalian) Co., Ltd). Approximately, 50 ng of template DNA was used for PCR amplification with SP (0.5 lmol/L) and AP (0.5 lmol/L) in a 50 lL reaction volume, 5u Ex-Taq (Takara Biotechnology (Dalian) Co., Ltd). The cycling program was as follows: 94 °C for 5 min; 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; and extended reaction at 72 °C for 5 min. The clear bands of the remaining PCR products were gel purified and sequenced (Sagon, Shanghai, China) directly by using the primer of either SP or DQW. The results were analyzed by BLAST in NCBI.
Results Selection of the Primers of rRNA for Bacterial Identification Genomic DNA of 23 strains bacteria of 18 species was used as templates for PCR walking to get sequences about rRNA. 357R primer was used to get upstream sequence of 16S rRNA, 474R primer was used to get intergenic sequence of 16S-23S rRNA, and 474F primer was used to get sequence of 23S rRNA. The results (Fig. 2; Table 2) prove that one-step PCR walking method can be used for different primers and bacteria. The sequences obtained from primer 357R of the 23 bacteria show that only one species of bacteria was the highest homologous, and can be used for bacterial identification. Identification of Different Bacteria The primer 357R was used to obtain upstream sequence of 16S rRNA. Using the primers of EU27F and 1492R, to obtain the sequence of 16S rRNA. The results of
Fig. 1 Basic principle of the AP gene-walking PCR. The known sequence stretch is shown as solid line, the unknown sequence stretch is shown as dotted line, SP is shown as black arrow, and AP is shown as black and white arrow. After 35 cycles, the target sequences were the products of the double primers of SP and AP; the nontarget sequences were the products of a single prime of SP or AP
123
488
H. Zhang et al.: A Simple One-Step PCR Walking Method
Fig. 2 Walking PCRs of different bacterial rRNA. Lane M: DL2, 000 DNA Marker; Lanes 1–3:357R, 474F, 474R. The bands shown by arrow were purified and sequenced
BLAST are shown in Table 3. For the BLAST analysis of the sequence upstream 16S rRNA, the bacterium with the highest score is corresponding to the bacterium of detection, and is significantly different from the other bacteria with the second highest score. All 41 selected strains of 31 species can be identified at the species level.
Discussion Compared with the ordinary walking PCR methods, the one-step PCR walking in this study has the advantages of convenient operation. Furthermore, the higher annealing temperature in the entire PCR procedure ensures the specificity and accuracy of the amplification. This is due to the fact that the AP primer consists of three parts (50 –30 ): 1.
The DQW primer, which was used as sequencing primer, can increase the pairing length of the APs to amplify the target sequence in the first round; DQW also can help forming panhandle structure
123
between primers of AP to inhibit the nonspecific amplification. 2. Six bases of degenerate sequence add more complementary sites to increasing annealing temperature. 3. Six bases of a specific sequence have high G/C % which can increase annealing temperature. As sequences of rRNA genes are obtained the universality of bacterial sequencing primers, and the variability of amplified sequences is achieved. It can also solve the problem that the mixed PCR products of multiple gene copies cannot be directly sequenced. The resolution of bacterial identification by using the primer 357R was increased to the species level. In practical applications, we also found that some bacteria sequences do not contain the corresponding upstream sequences of the 16S rRNA genes deposited in public databases. This leads to difficulties in identifying highly related sequences by BLAST analysis as often several sequences show the highest BLAST scores. With the speed at which genomes of bacterial strains are sequences it will one day be unnecessary to do PCR at all as relationships will be determined by IT programs.
Streptococcus thermophilus St-body 3
Streptococcus thermophilus TA40(St01)
Lactobacillus delbrueckii ssp.bulgaricus LB-12
Lactobacillus delbrueckii ssp.bulgaricus LB340(LB340)
Lactobacillus casei 01(LC01)
Lactobacillus casei Shirota(LCYA)
Lactobacillus casei LC2W(LC2W)
357R
Lactobacillus casei 431(LC431)
DQW-5
Sequencing primer
DQW-5 DQW-5
474R 357R
100 474R
474R
KC161281 (768)
99.9
100 KC161271 (748)
474F
100
474F
KC161292 (624)
KC161280 (548)
The first 357 nt of 16S rRNA (301 bp)
357R
474R
474R 357R
100 99.3
100 KC161270 (283)
474F
99.9
99.6
The first 357 nt of 16S rRNA (285 bp)
KC161291 (717)
KC161279 (811)
474F
357R
474R
474R 357R
100 99.9
474F
KC161269 (756)
The first 357 nt of 16S rRNA (83 bp) 474F
KC161278 (275) KC161297 (605)
97.5 99.2
99.3
KC161267 (452)
474F
474F
98.5
100 100
KC161296 (600)
KC161277 (735)
The first 357 nt of 16S rRNA (74 bp)
357R
DQW-5
474R 357R
100
KC161266 (713)
DQW-5
100
474F
KC161261 (695) No
DQW-5
The first 357 nt of 16S rRNA
357R
474R
474R
100
100
KC161276 (856)
No KC161265 (688)
99.7
474F
KC161260 (664)
99.3
100
100 No
99.9
100
No
95.5
Identities of the target bacteria (%)
474F
DQW-5
KC161275 (544)
KC161264 (718)
KC161289 (578)
KC161274 (786 )
KC161263 (715)
KC161259 (779)
Accession number (length) (bp)
The first 357 nt of 16S rRNA
357R
DQW-5 DQW-5
474F 474R
357R DQW-5 The first 357 nt of 16S rRNA
DQW-5 DQW-5
474F 474R
The first 357 nt of 16S rRNA
Walking primer
Bacterial name
Table 2 Selection of the primers about rRNA for bacterial identification
99.2 (Streptococcus salivarius)
99.9 (Streptococcus salivarius)
99.7 (Streptococcus salivarius)
94.2 (Streptococcus salivarius)
98.5 (Streptococcus salivarius)
100 (Streptococcus salivarius)
99.6 (Streptococcus salivarius)
98.9 (Streptococcus salivarius)
83.5 (Lactobacillus helveticus)
91.7 (Lactobacillus helveticus)
91.6 (Lactobacillus helveticus)
75.3 (Lactobacillus helveticus) 13.1 (Lactobacillus kefiranofaciens)
92.5 (Lactobacillus helveticus)
100 (Lactobacillus acidophilus)
13.7 (Lactobacillus kefiranofaciens)
98 (Lactobacillus rhamnosus)
98.6 (Lactobacillus rhamnosus)
No
82.3 (Lactobacillus rhamnosus)
97.9 (Lactobacillus rhamnosus)
98.5 (Lactobacillus rhamnosus)
No
80.7 (Lactobacillus rhamnosus)
96.9 (Lactobacillus rhamnosus)
98.6 (Lactobacillus rhamnosus)
94.5 (Lactobacillus rhamnosus) No
97.8 (Lactobacillus rhamnosus)
96.8 (Lactobacillus rhamnosus)
No
84.5 (Lactobacillus rhamnosus)
Identities of the nontarget bacteria (%)
H. Zhang et al.: A Simple One-Step PCR Walking Method 489
123
123
Corynebacterium glutamicum ACCC16522
Bifidobacterium longum subsp. longum CICC 21717
Bacillus pumilus CMCC63202
Bacillus megaterium ACCC11011
Bacillus cereus ATCCC1220
Lactobacillus.acidophilus NCFM(NCFM)
Escherichia coli DH5a
KC161294 (685)
KC161282 (696)
357R
357R
KC768812 (381)
99.0
100
357R
KC768811 (375)
KC768839 (630)
474F 474R
474F 474R
KC768840 (454)
\200 bp (131)
The first 357 nt of 16S rRNA(242 bp)
357R
474R
474R
100
100
99.6
94.7
99.8
100
KC768830 (659)
100
99.5
474F
KC415589 (804)
\200 bp (187)
The first 357 nt of 16S rRNA(283 bp)
357R
474R
474F
357R
474R
99.7 99.8
The first 357 nt of 16S rRNA(297 bp) 474F 474F KC768832 (420)
97.9
\200 bp (141)
98.1
100
\200 bp (150)
99.6
KC768816 (424)
357R
474R
474R 357R
474F
474F
The first 357 nt of 16S rRNA(245 bp)
99.6
100
KC768834 (568)
KC768827 (909)
474F 474R
474F
97.9
474R
KC415592 (426) 97.3
357R
KC161284 (671)
The first 357 nt of 16S rRNA(188 bp)
357R
DQW-5
474R
99.6
99.8
KC161273 (427)
100
100
100 99.8
474F
KC161295 (743)
\200 bp (127) KC161283 (573)
The first 357 nt of 16S rRNA(299 bp)
357R
474F 474R
100
100
100
474F
357R
474F 474R
The first 357 nt of 16S rRNA(311 bp)
DQW-5
474R
357R
474R
KC161272 (681)
99.9
100
474F
KC161293 (748)
Identities of the target bacteria (%)
474F
357R
Accession number (length) (bp)
100
357R
Lactobacillus Plantarum ST-III(ST-III)
Sequencing primer
The first 357 nt of 16S rRNA (318)
Walking primer
Bacterial name
Table 2 continued
No
29.0 (Corynebacterium urealyticum)
85.1 (Rhodococcus globerulus)
54.9 (Rhodococcus globerulus)
99.7 (Bifidobacterium longum subsp. infantis)
100 (Bifidobacterium longum subsp. infantis)
100 (Bifidobacterium longum subsp. Infantis)
75.6 (Bifidobacterium breve)
93.6 (Bacillus atrophaeus)
97.3 (Bacillus safensis) 99.5 (Bacillus subtilis subsp)
68.1 (Bacillus safensis)
55.3 (Bacillus cereus subsp. cytotoxis)
95.3 (Bacillus thuringiensis serovar finitimus)
98.0 (Bacillus aryabhattai)
63.0 (Bacillus aryabhattai)
99.6 (Bacillus thuringiensis)
100 (Bacillus thuringiensis)
97.3 (Bacillus weihenstephanensis)
93.7 (Bacillus thuringiensis serovar finitimus)
97.2 (Lactobacillus amylovorus)
98.1 (Lactobacillus crispatus)
97.0 (Lactobacillus helveticus)
91.5 (Lactobacillus amylovorus)
100 (Shigella sonnei) 98.8 (Shigella sonnei)
99.7 (Shigella dysenteriae)
98.4 (Shigella flexneri)
92.5 (Lactobacillus pentosus)
99.3 (Lactobacillus pentosus)
100 (Lactobacillus pentosus)
49.3 (Lactobacillus pentosus)
Identities of the nontarget bacteria (%)
490 H. Zhang et al.: A Simple One-Step PCR Walking Method
Shigella sonnei CMCC51334
Shigella flexneri CCAM 090021
Salmonella Typhimurium ATCC14023
Lysinibacillus sphaericus ACCC11096
Lactococcus lactis subsp. lactis Il1403
Enterobacter cloacae ATCC13047
357R
KC768814 (329)
KC768838 (848)
357R
KC768831 (452) KC768817 (712)
KC768833 (415)
474R
100
KC768819 (705)
KC768835 (441)
474F 474R
474F 474R
KC768843 (604)
\200 bp (132)
The first 357 nt of 16S rRNA (300 bp)
357R
474R
100
100
100
100
100
357R
474R
99.3
100
100
100
100
100
100 100
KC415591 (539)
KC768841 (602)
\200 bp (128)
100 100
The first 357 nt of 16S rRNA (303 bp) 474F 474F \200 bp (127)
357R
474R 357R
474F
474F
The first 357 nt of 16S rRNA (283 bp)
357R
474F 474R
474F 474R
The first 357 nt of 16S rRNA (295 bp)
357R
474R
474R
100
100
KC768829 (886)
100
100
100 97.6
100
474F
KC415597 (764)
\200 bp (132) KC768842 (586)
The first 357 nt of 16S rRNA (311 bp)
357R
474F 474R
99.5 94.0
474F
357R
474F 474R
The first 357 nt of 16S rRNA (292 bp)
KC768826 (699)
KC768836 (611)
474R 357R
474R 357R
100
KC768828 (672)
99.3
474F
KC415588 (731)
Identities of the target bacteria (%)
474F
DQW-5
Accession number (length) (bp)
No
357R
Enterobacter aerogenes ATCC13048
Sequencing primer
The first 357 nt of 16S rRNA
Walking primer
Bacterial name
Table 2 continued
99.5 (Escherichia coli)
100 (Shigella sonnei)
100 (Escherichia coli)
99.3 (Escherichia coli)
99.5 (Shigella boydii)
99.7 (Escherichia coli) 100 (Escherichia coli)
98.1 (Escherichia coli)
99.5 (Salmonella typhimurium)
98.4 (Pectobacterium carotovorum subsp)
99.6 (Salmonella paratyphi)
95.5 (Salmonella bongori)
92.5 (Solibacillus silvestris)
97.3 (Solibacillus silvestris)
99.3 (Brevibacillus laterosporus)
89.1 (Brevibacillus laterosporus)
99.9 (Lactococcus lactis subsp. cremoris)
100 (Lactococcus lactis subsp. cremoris)
99.4 (Streptococcus lactis)
65.3 (Lactococcus lactis subsp. cremori)
100 (Pantoea endophytica) 96.2 (Klebsiella pneumoniae subsp. pneumoni)
100 (Escherichia coli)
93.0 (Escherichia coli)
90.3 (Enterococcus hirae)
98.4 (Enterococcus faecium)
No
83.0 (Klebsiella oxytoca)
Identities of the nontarget bacteria (%)
H. Zhang et al.: A Simple One-Step PCR Walking Method 491
123
123 100
KC768816 (424) KC768811 (375) KC768826 (699) KC161294 (685) KC768813 (576)
Corynebacterium glutamicum ACCC16522
Enterobacter cloacae ATCC13047
Escherichia coli DH5a
Kocuria rhizophila DC2201
KC161289 (578)
KC768812 (381)
Bacillus megaterium ACCC11011
Bacillus pumilus CMCC63202
KC161260 (664)
KC415589 (804)
Bifidobacterium longum subsp. longum CICC 21717
Lactobacillus casei LC2 W
KC415597 (764)
Lactococcus lactis subsp. lactis Il1403
Lactobacillus casei Shirota
100
KC415598 (719)
Lactobacillus casei ATCC 334
KC161261 (695)
KC415596 (699)
Lactobacillus casei str. Zhang
KC161259 (779)
KC415595 (764)
Lactobacillus plantarum WCFS1
Lactobacillus casei 01
KC415594 (761)
Streptococcus thermophilus LMD-9
Lactobacillus casei 431
100
KC415593 (700)
Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293
99.3
99.7
100
95.5
100
94.0
94.7
98.1
99.0
99.5
100
99.7
100
100
100
99.9
99.8
KC415588 (731) KC415599 (571)
Enterobacter aerogenes ATCC13048
99.3
Enterococcus faecalis ACCC10699
KC415590 (735) KC415591 (539)
Escherichia coli ATCC25922
Shigella flexneri CCAM 090021
99.2 97.9
KC415587 (766) KC415592 (426)
Bacillus licheniformis ACCC02002
Bacillus cereus ATCCC1220
99.0
KC415586 (491)
80.7 (Lactobacillus rhamnosus)
94.5 (Lactobacillus rhamnosus)
84.5 (Lactobacillus rhamnosus)
82.3 (Lactobacillus rhamnosus)
36.5 (Arthrobacter globiformis)
98.4 (Shigella flexneri)
93.1 (Escherichia coli)
54.9 (Rhodococcus globerulus)
68.2 (Bacillus safensis strain)
63.0 (Bacillus aryabhattai)
75.6 (Bifidobacterium breve)
65.3 (Lactococcus lactis subsp. cremori)
79.4 (Lactobacillus rhamnosus)
KC768796 (1,406)
KC768795 (1,399)
KC768794 (1,365)
KC768797 (1,310)
KC768807 (1,360)
KC768803 (1,356)
KC768786 (1,369)
KC768805 (1,355)
KC768810 (1,400)
KC768806 (1,402)
KC429787 (1,359)
KC429785 (1,411)
KC429784 (1,400)
KC429783 (1,425)
KC429782 (1,420)
85.4 (Lactobacillus rhamnosus)
KC429781 (1,381)
100 (Lactobacillus pentosus)a
KC429780 (1,402)
KC429779 (1,402)
KC429778 (1,376)
KC429777 (1,353)
KC429776 (1,355)
KC429775 (1,396)
KC429774 (1,393)
KC429773 (1,397)
80.7 (Streptococcus salivarius)
95.5 (Leuconostoc carnosum)
87.2 (Enterococcus hirae)
83.0 (Klebsiella oxytoca)
98.1 (Escherichia coli)
97.4 (Shigella flexneri)
93.7 (Bacillus thuringiensis serovar finitimus)
63.8 (Bacillus subtilis)
91.2 (Bacillus atrophaeus)
Accession number (length) (bp)
Identities of the nontarget bacteria (%)
Accession number (length) (bp)
Identities of the target bacteria (%)
Sequence of 16S rRNA
Sequence of 357R
Bacillus subtilis CMCC63501
Bacterial name
Table 3 BLAST analysis of different bacteria
100
100
100
99.9
100
100
100
100
99.9
100
100
100
100
100
100
100
100
100
100
100
100
100
99.0
100
Identities of the target bacteria (%)
100 (Lactobacillus paracasei)
100 (Lactobacillus paracasei)
100 (Lactobacillus paracasei)
99.9 (Lactobacillus paracasei)
99.8 (Arthrobacter globiformis)
100 (Shigella flexneri)
99.9 (Enterobacter asburiae)
99.4 (Corynebacterium acetoacidophilum)
99.9 (Bacillus safensis)
100 (Bacillus aryabhattai)
100 (Bifidobacterium longum subsp. Infantis)
100 (Lactobacillus paracasei;Lactobacillus rhamnosus) 99.9 (Lactococcus garvieae)
100 (Lactobacillus paracasei;Lactobacillus rhamnosus)
100 (Enterococcus durans)
99.8 (Streptococcus salivarius)
99.9 (Lactobacillus delbrueckii subsp. bulgaricus)
100 (Enterococcus faecium)
99.9 (Pantoea sp.)
100 (Escherichia coli)
99.9 (Shigella flexneri)
100 (Bacillus anthracis)
98.7 (Bacillus aerius)
100 (Bacillus tequilensis;Bacillus methylotrophicus)
Identities of the nontarget bacteria (%)
492 H. Zhang et al.: A Simple One-Step PCR Walking Method
KC768820 (711) KC768819 (705) KC768822 (445) KC161292 (624) KC161291 (717) KC768823 (342) KC768824 (805) KC768825 (644)
Shigella dysenteriae CMCC51335
Shigella sonnei CMCC51334
Staphylococcus aureus ATCC8095
Streptococcus thermophilus St-body 3
Streptococcus thermophilus TA40
Vibrio cholerae SJTU32001
Vibrio parahaemolyticus ATCC17802
Vibrio vulnficus ATCC27562
98.8
99.3
100
99.9
100
100
100
99.2
100
99.6
95.3
99.5
100
100
100
98.5
99.2
78.1 (Vibrio cholerae)
59.6 (Vibrio harveyi)
86.8 (Vibrio mimicus strain)
98.9 (Streptococcus salivarius)
94.2 (Streptococcus salivarius)
95.3 (Staphylococcus epidermidis)
99.3 (Escherichia coli)
98.3 (Escherichia coli)
95.5 (Salmonella bongori)
90.9 (Salmonella bongori)
93.4 (Pseudomonas brassicacearum subsp)
72.5 (Pseudomonas stutzeri)
89.1 (Brevibacillus laterosporus)
91.5 (Lactobacillus amylovorus)
49.3 (Lactobacillus pentosus)
13.7 (Lactobacillus kefiranofaciens)
13.1 (Lactobacillus kefiranofaciens)
KC768793 (1,361)
KC768792 (1,368)
KC768791 (1.363)
KC768800 (1,372)
KC768801 (1,374)
KC768790 (1,376)
KC768787 (1,362)
KC768788 (1,369)
KC768784 (1,367)
KC768785 (1,360)
KC768809 (1,386)
KC768789 (1,317)
KC768808 (1,377)
KC768804 (1,394)
KC768802 (1,400)
KC768798 (1,374)
KC768799 (1,381)
Lactobacillus pentosus MP-10 draft has no complete sequence in NCBI; the sequence of BLAST is too short (378/764)
KC768817 (712)
Salmonella Typhimurium ATCC14023
a
KC768818 (689)
Salmonella Heidelberg ATCC13311
KC768814 (329)
Lysinibacillus sphaericus ACCC11096 KC768821 (799)
KC161295 (743)
Lactobacillus.acidophilus NCFM
KC768815 (682)
KC161293 (748)
Lactobacillus Plantarum ST-III
Pseudomonas aeruginosa CDCB32116
KC161296 (600)
Lactobacillus delbrueckii ssp.bulgaricus LB340
Pseudomonas fluorescens AS1.823
KC161297 (605)
Accession number (length) (bp)
Identities of the nontarget bacteria (%)
Accession number (length) (bp)
Identities of the target bacteria (%)
Sequence of 16S rRNA
Sequence of 357R
Lactobacillus delbrueckii ssp.bulgaricus LB-12
Bacterial name
Table 3 continued
100
99.9
100
100
100
100
100
100
100
100
99.9
100
100
100
99.9
100
100
Identities of the target bacteria (%)
97.3 (Vibrio aestuarianus)
99.9 (Vibrio alginolyticus)
99.9 (Vibrio mimicus)
99.8 (Streptococcus salivarius)
99.8 (Streptococcus salivarius)
98.8 (Staphylococcus epidermidis)
99.9 (Escherichia coli)
99.7 (Escherichia coli)
99.1 (Salmonella paratyphi)
99.9 (Salmonella choleraesuis)
99.9 (Pseudomonas chlororaphis subsp)
99.9 (Rhodococcus erythropolis)
100 (Lysinibacillus fusiformis)
99.6 (Lactobacillus kitasatonis)
99.9 (Lactobacillus pentosus)
99.3 (Lactobacillus leichmannii)
99.7 (Lactobacillus delbrueckii subsp. sunkii)
Identities of the nontarget bacteria (%)
H. Zhang et al.: A Simple One-Step PCR Walking Method 493
123
494 Acknowledgments This work was supported by the Major Project of Chinese National Programs for Fundamental Research and Development (973 Program) (grant no. 2012CB723706).
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