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SCCmec同定法

PCRによるSCCmecの同定法

 

MRSA株のSCCmecのタイプはPCRで特定の遺伝子や遺伝子複合体を検出して決定するのが一般的です。以下のような方法がこれまで報告されています。

 

I. SCCmecタイプの同定

1.  ccr type を同定する PCR

ccr遺伝子複合体の同定はその中心であるccr遺伝子の同定で代行しています。

(1) ccr1, ccr2, ccr3の検出Ito et al. 2001, Okuma et al. 2002

ccrB1, ccrB2, ccrB3に共通な一つのプライマーとccrA1, ccrA2, ccrA3に特有なそれぞれのプライマーを用いてccrAの違いにより、ccr1, ccr2, ccr3を区別して検出するmultiplexPCR.

(2) ccr1, ccr2, ccr3, ccr5ccrC)の検出 (Zhang et al. 2005)。

(1)のプライマーの組み合わせにccrCの検出を加えたもの

(3) ccr1, ccr2, ccr3, ccr4, ccr5 (ccrC)の検出 (Kondo et al. submitted)。

(1)のプライマーの組み合わせに、ccr4ccrCの検出を加えたもの。

 

2.mec遺伝子複合体のクラスを同定するPCR

mecAに加えてmec遺伝子複合体を構成する遺伝子(mecI, mecR1などの制御遺伝子及び近傍の挿入配列IS431, IS1272)を検出する。

(1) 遺伝子及び遺伝子複合体をそれぞれ増幅する従来法(Okuma et al.2002. Hisata et al. 2005. Chongtracool et al. 2006)

(2) classAmec, class B mecを同時に検出するMultiplex PCRZhang et al. 2005

(3) classAmec, classBmec, classCmecを同時に検出するMultiplex PCR (Kondo et al. submitted)

 

PCRの結果同定されたccr typeとmec classの組み合わせからSCCmecのタイプを判定します。

ccr mec combination of ccr and mec SCCmec タイプ
type 1 class B 1B Type I
type 2 class A 2A Type II
type 3 class A 3A Type III
type 2 class B 2B Type IV
type 5 class C 5C Type V
type 4 class B 4B Type VI

 

3. J領域の構造の違いに基づくサブタイピング。

SCCmecのJ1領域の構造の違いにより分類する方法、及びJ2-3領域に挿入された薬剤耐性プラスミドやトランスポゾンの有無により分類する方法がこれまでに報告されています。

(1). Multiplex PCROliveira et al. 2001

最初に報告されたMultiplex PCRでtype I.1, type II.1 SCCmecの J1領域に存在するORF、薬剤耐性プラスミドpT181, pUB110の存在、SCCmercuryの存在を同時に検出できる。

(2). それぞれに特異的なprimer pairを用いる方法Okuma et al.2002. Ma et al. 2005. Hisata et al. 2005

Type-II.1, type-II.2, SCCmec, type-IV.1, type-IV.2, type-IV.3, type-IV.4などの SCCmec のJ1領域特有のORFを検出する。

(3) Multiplex PCRZhang et al. 2005

type-I.1, type-II.1, type-IV.1, type-IV.2, type-IV.4 SCCmecのJ1領域特有のORFを検出できる。Type-III.1 SCCmecを検出するとされているプライマーセットはSCCmercury上に、type-IV.3 SCCmecを検出するとされているプライマーセットはSCCmecの外側の外来領域上にある点に注意する必要。

(4) Multiplex PCR #3 (Kondo et al. submitted)

type I.1, type IV.1, type IV.2, type IV.3, type IV.4 SCCmecのJ1領域特有のORFを検出する。

(5) Muliplex PCR #4 (Kondo et al. submitted)

type II.1, type II.2, type II.3, type II.4, type III.1, type V.1 SCCmecのJ1領域特有のORFを検出する。

(6) Multiplex PCR#5 ( Kondo et al. submitted)

type II, type-III SCCmecのJ2領域とトランスポソンTn554及びYTn554の存在を確認できる。

(7) Multiplex PCR#6 ( Kondo et al. submitted)

mecA遺伝子の下流に挿入されているpUB110とpT181の存在を検出できる。

 

II. PCRに用いるプライマーセット

それぞれのPCRに用いる プライマーセット及び反応条件

1. ccr type を同定する PCR

目的 プライマー (以前の名称) 塩基配列 (5'-3') プライマーを設計した遺伝子 期待されるDNA断片のサイズ (増幅される領域 ) 反応条件 コメント
(1) ccr1, ccr2, ccr3の検出(Ito et al. 2001) ßc (ß2) ATTGCCTTGATAATAGCCITCT ccrB1, ccrB2, and ccrB3 A I, は inosine. 本PCRは三つのccrBに共通したpプライマー ßc とccrAの三つのタイプそれぞれに反応するプライマーa1 (ccrA1), a2 (ccrA2),  a3 (ccrA3)を用いて、ccrAのタイプの違いによりタイピングする方法である。
α1 (α2) AACCTATATCATCAATCAGTACGT ccrA1 695 bp (ccrA1-ccrB1)
α2 (α3) TAAAGGCATCAATGCACAAACACT ccrA2 937 bp (ccrA2-ccrB2)
α3 (α4) AGCTCAAAAGCAAGCAATAGAAT ccrA3 1791 bp (ccrA3-ccrB3)
(2) ccr1, ccr2, ccr3, ccr5(ccrC)の検出  (Zhang et al. 2005) ccrAB-ß2 the same as ßc(ß2) ccrB1, ccrB2, and ccrB3 C
ccrAB-2 the same as     α1 (α2) ccrA1 695 bp (ccrA1-ccrB1)
ccrAB-3 the same as     α2 (α3) ccrA2 937 (ccrA2-ccrB2)
ccrAB-4 the same as     α3 (α4) ccrA3 1791 bp (ccrA3-ccrB3)
ccrC-F ATGAATTCAAAGAGCATGGC ccrC 336 bp (ccrC)
ccrC-R GATTTAGAATTGTCGTGATTGC ccrC
(3) ccr1, ccr2, ccr3, ccr4, ccr5 (ccrC)の検出  (Kondo et al. submitted) mA1 TGCTATCCACCCTCAAACAGG mecA 286 bp (mecA) D
mA2 AACGTTGTAACCACCCCAAGA mecA
α1 AACCTATATCATCAATCAGTACGT ccrA1 695 bp (ccrA1-ccrB1)
α2 TAAAGGCATCAATGCACAAACACT ccrA2 937 bp (ccrA2-ccrB2)
α3 AGCTCAAAAGCAAGCAATAGAAT ccrA3 1791 bp (ccrA3-ccrB3)
βc ATTGCCTTGATAATAGCCITCT ccrB1, ccrB2, and ccrB3
α4.2 GTATCAATGCACCAGAACTT ccrA4 1287 bp (ccrA4-ccrB4)
β4.2 TTGCGACTCTCTTGGCGTTT ccrB4
γR CCTTTATAGACTGGATTATTCAAAATAT ccrC D
γF CGTCTATTACAAGATGTTAAGGATAAT ccrC

2.mec遺伝子複合体のクラスを同定するPCR

目的 プライマー (以前の名称) 塩基配列 (5'-3') プライマーを設計した遺伝子 期待されるDNA断片のサイズ (増幅される領域 ) 反応条件 コメント
(1) 遺伝子及び遺伝子複合体をそれぞれ増幅する従来法(Okuma et al.2002. Hisata et al. 2005. Chongtracool et al. 2006)
mecA mA1 TGCTATCCACCCTCAAACAGG mecA 286 bp  (mecA) A
mA2 AACGTTGTAACCACCCCAAGA mecA
class A mec mI4 CAAGTGAATTGAAACCGCCT mecI 187bp (mecI for class A mec) A
mI3 CAAAAGGACTGGACTGGAGTCCAAA mecI
mcR2 CGCTCAGAAATTTGTTGTGC mecR1(PB) 319 bp (PB domein of mecR1 for class A mec)
mcR5 CAGGGAATGAAAATTATTGGA mecR1(PB)
class A mec mI4 CAAGTGAATTGAAACCGCCT mecI 1920 bp  (mecI-mecR1 for class A mec) A
mcR3 GTCTCCACGTTAATTCCATT mecR1
class B mec IS5 AACGCCACTCATAACATATGGAA IS1272 1996 bp, IS5 (mecA-IS1272 for class B mec) A
mA6 TATACCAAACCCGACAAC mecA
class C mec IS2 (iS-2) TGAGGTTATTCAGATATTTCGATGT IS431mec 2072 bp, IS2 (mecA-IS431 for class C mec) A
mA2 AACGTTGTAACCACCCCAAGA mecA
(2) classAmec, class B mecを同時に検出するMultiplex PCR(Zhang et al. 2005) class C mec CCCTTTTTATACAATCTCGTT mecI 146 bp (mecI for class A mec) C
mecI-R ATATCATCTGCAGAATGGG mecI
IS1272-F TATTTTTGGGTTTCACTCGG IS1272 1305  bp (IS1272-mecR1 for class B mec)
mecR1-R CTCCACGTTAATTCCATTAATACC mecR1
(3) classAmec, classBmec, classCmecを同時に検出するMultiplex PCR (Kondo et al. submitted) mI6 CATAACTTCCCATTCTGCAGATG mecI 1963  bp (mecA-mecI for class A mec) E
IS7 ATGCTTAATGATAGCATCCGAATG IS1272 in the upstream of mecA 2827 bp (mecA-IS1272 for class B mec)
IS2(iS-2) TGAGGTTATTCAGATATTTCGATGT IS431in the upstream of mecA 804  bp (mecA-IS431 for class C mec)
mA7 ATATACCAAACCCGACAACTACA mecA

3. J領域の構造の違いに基づくサブタイピング

目的 プライマー (以前の名称) 塩基配列 (5'-3') プライマーを設計した遺伝子 期待されるDNA断片のサイズ (増幅される領域 ) 反応条件 コメント
(1). Multiplex PCR ( Oriveira et al. 2001) CIF2 F2 TTCGAGTTGCTGATGAAGAAGG I 495 B Locus A
CIF2 R2 ATTTACCACAAGGACTACCAGC
KDP F1 AATCATCTGCCATTGGTGATGC II 284 Locus B
KDP R1 CGAATGAAGTGAAAGAAAGTGG
MECI P2 ATCAAGACTTGCATTCAGGC II, III 209 Locus C
MECI P3 GCGGTTTCAATTCACTTGTC
DCS F2 CATCCTATGATAGCTTGGTC I, II, IV 342 Locus D
DCS R1 CTAAATCATAGCCATGACCG
RIF4 F3 GTGATTGTTCGAGATATGTGG III 243 Locus E
RIF4 R9 CGCTTTATCTGTATCTATCGC
RIF5 F10 TTCTTAAGTACACGCTGAATCG III 414 Locus F
RIF5 R13 GTCACAGTAATTCCATCAATGC
IS431 P4 CAGGTCTCTTCAGATCTACG 381 Locus G
pUB110 R1 GAGCCATAAACACCAATAGCC
IS431 P4 CAGGTCTCTTCAGATCTACG 303 Locus H
pT181 R1 GAAGAATGGGGAAAGCTTCAC
MECA P4 TCCAGATTACAACTTCACCAGG Internal control 162 mecA
MECA P7 CCACTTCATATCTTGTAACG
(2). それぞれに特異的なprimer pairを用いる方法(Okuma et al.2002. Ma et al. 2005. Hisata et al. 2005) 2a1 ATGTCAGAGCTTTCTAACTTAGTCA IIa 456 A
2a2 TGAAAATGAAAGCCGTGCCG
2b1 AGCAATTTTTTCTCCTTCTGCTA II b 846 A
2b2 TTATTAGATCAAGAGCCAAGTG
4a1 TTTGAATGCCCTCCATGAATAAAAT IVa 458 A
4a2 AGAAAAGATAGAAGTTCGAAAGA
4b1 AGTACATTTTATCTTTGCGTA IVb 994 A
4b2 AGTCATCTTCAATATCGAGAAAGTA
4c1 TCTATTCAATCGTTCTCGTATTT IVc 678 A
4c2 TCGTTGTCATTTAATTCTGAACT
4d1 TTTGAGAGTCCGTCATTATTTCTT IVd 1010 A
4d2 AGAATGTGGTTATAAGATAGCTA
(3) Multiplex PCR (Zhang et al. 2005) Type I-F GCTTTAAAGAGTGTCGTTACAGG SCCmec I 613 C *type-III-F、type-III-Rの組み合わせで増幅される領域はSCCmercury 上にある。type-IVc-R, type-Ivc-R の組み合わせで増幅される領域はtype-IV SCCmecに隣接した領域(IE25923)である。
Type I-R GTTCTCTCATAGTATGACGTCC
Type II-F CGTTGAAGATGATGAAGCG SCCmec II 398
Type II-R CGAAATCAATGGTTAATGGACC
Type III-F CCATATTGTGTACGATGCG SCCmec III 280
Type III-R CCTTAGTTGTCGTAACAGATCG
Type IVa-F GCCTTATTCGAAGAAACCG SCCmec IVa 776
Type IVa-R CTACTCTTCTGAAAAGCGTCG
Type IVb-F TCTGGAATTACTTCAGCTGC SCCmec IVb 493
Type IVb-R AAACAATATTGCTCTCCCTC
Type IVc-F ACAATATTTGTATTATCGGAGAGC SCCmec IVc 200
Type IVc-R TTGGTATGAGGTATTGCTGG
Type IVd-F5 CTCAAAATACGGACCCCAATACA SCCmec IVd 881
Type IVd-R6 TGCTCCAGTAATTGCTAAAG
Type V-F GAACATTGTTACTTAAATGAGCG SCCmec V 325
Type V-R TGAAAGTTGTACCCTTGACACC
(4) Multiplex PCR #3  (Kondo et al. submitted) 1a3 TTTAGGAGGTAATCTCCTTGATG Type-I.1 SCCmec 154 bp (E007 in Type-I.1 SCCmec) E
1a4 TTTTGCGTTTGCATCTCTACC
4al TTTGAATGCCCTCCATGAATAAAAT Type  IV.1 (IVa) SCCmec 458 bp (CQ002  in Type  IV.1 (IVa) SCCmec  )
4a3 AGAAAAGATAGAAGTTCGAAAGA
4b3 AACCAACAGTGGTTACAGCTT Type IV.2 (IVb) SCCmec 726 bp ( M001 in Type IV.2 (IVb) SCCmec)
4b4 CGGATTTTAGACTCATCACCAT
4c4 AGGAAATCGATGTCATTATAA Type IV.3(IVc) SCCmec 259  bp(CR008 in Type IV.3(IVc) SCCmec
4c5 ATCCATTTCTCAGGAGTTAG
4d3 AATTCACCCGTACCTGAGAA Type IV.4(IVd)  SCCmec 1242 bp (CD002 in Type IV.4(IVd)  SCCmec)
4d4 AGAATGTGGTTATAAGATAGCTA
(5) Muliplex PCR #4 (Kondo et al. submitted) kdpB1 GATTACTTCAGAACCAGGTCAT KdpB in  Type II.1 SCCmec 287  bp (KdpB in  Type II.1 SCCmec) E
kdpB2 TAAACTGTGTCACACGATCCAT
2b3 GCTCTAAAAGTTGGATATGCG S01 in Type II.2 SCCmec 1518 bp  (S01 in Type II.2 SCCmec  )
2b4 TGGATTGAATCGACTAGAATCG
4b3 AACCAACAGTGGTTACAGCTT IIE03 in type II.3 (IIE) SCCmec and M001 in Type IV.2 (IVb) SCCmec 726 bp ( IIE03 in type II.3 (IIE) SCCmec and M001 in Type IV.2 (IVb) SCCmec)
4b4 CGGATTTTAGACTCATCACCAT
II4-3 GTACCGCTGAATATTGATAGTGAT RN06 in Type-II.4 SCCmec 2003 bp (RN06 in Type-II.4 SCCmec)
II4-1 ACTCTAATCCTAATCACCGAAC
3a1 ATGGCTTCAGCATCAATGAG Z004 in type-III.1 SCCmec 503 bp  (Z004 in type-III.1 SCCmec)
3a2 ATATCCTTCAAGCGCGTTTC
5a1 ACCTACAGCCATTGCATTATG V024 in type-V SCCmec 1159 bp  (V024 in type-V SCCmec)
5a2 TGTATACATTTCGCCACTAGCT
(6) Multiplex PCR#5 ( Kondo et al. submitted) ermA1 TGAAACAATTTGTAACTATTGA ermA 2756 bp (ermA-ORFs (CN030 and CZ021) in J2 region of type-II.1 and III.1 SCCmec) E
cad4 ATTGCGATTCTTTCCGATATGG cadB 1540 bp (cadB- ORFs (CN030 and CZ021)  in J2 region of type-II.1 and III.1 SCCmec)
mN5 TTGCTTCGGGACTTACCTCTAGT ORFs (CN030 and CZ021) in J2 region of type-II.1 and III.1 SCCmec
(7) Multiplex PCR#6 ( Kondo et al. submitted) ant1 CAGACCAATCAACATGGCACC ant(4)' in pUB110 4952 bp (mecA-ant(4)') F
pT181-2 AGGTTTATTGTCACTACAATTGA tetK in pT181 7406 bp (mecA-tetK)
mA1 TGCTATCCACCCTCAAACAGG mecA

III. 結果の記載

ccr and mec PCRにより検出されたJ領域 SCCmec type
1B type-I.1 specific ORF in J1 type-I.1 あるいはtype-Ia
2A type-II.1 specific ORF in J1 type-II.1 あるいはtype-IIa
2A type-II.2 specific ORF in J1 type-II.2 あるいはtype-IIb
2A type-II.3 specific ORF in J1 type II.3 あるいはtype IIc

 

PCR法

A.

The method at the early stage

All PCR reactions were carried out in a GeneAmp PCR system 9600 (Perkin Elmer, U. S. A.). The reactions were performed in a final volume of 100 ml and contained 10 ng of template DNA, two oligonucleotide primers (0.1 mM), 200 mM of each dNTP, 10 mM Tris-HCl, 50 mM KCl, 1.5mM MgCl2, 0.1% (w/v) gelatin and 2U of Taq DNA polymerase (Perkin Elmer, U. S. A). The PCR reaction consisted of 30 cycles of denaturation (94°C, 1 min), annealing (50°C, 1 min), and extension (72°C, 2 min).

The method now used at Department of Bacteriology, Juntendo University.

1. Prepare the pre-mixture and aliquot to each tube. Pre-mixture should contain the following components in a volume of 49 mL per aliquot: 5 mL 10 x commercial reaction buffer, 250 mM of each dNTP, 2-4 oligonucleotide primers (0.1 mM), Taq DNA polymerase (1 unit), and DEPC-H2O to adjust the final volume to 49 mL.

2. Add 1 mL of template DNA to each tube and subject to PCR.

3. The PCR conditions are: denaturation (94 °C, 1 min), 30 cycles of denaturation (94 °C, 1 min), annealing (50 °C, 30 sec or 1 min), and extension (72 °C, 2 min). Keep samples at 4 °C following PCR.

4. Mount the agarose gel in the electrophoresis tank, and add enough 1x TAE buffer to cover the gel.

5. Mix 4 mL sample from each PCR reaction with 1 mL loading buffer and load samples into the wells of the gel. The gel is usually run at high voltage (100V). Stop running at appropriate time. Usually, we stop the procedure when the bromophenol blue has run 2/3 of the gel length.

6. DNA fragments in the agarose gel are stained by soaking in 0.01% Ethidium Bromide solution for 20 min.

7. Take a photograph using transmitted UV light with Fas II (UV sample camera, Toyobo, Tokyo, Japan).

 

B. Multiplex PCR (Oliveira et al. 2001)

The multiplex PCR was performed in a 50-µl volume with the GeneAmp PCR kit (Applied Biosystems, Foster City, Calif.) containing the following: 1x PCR buffer II; 200 µM (each) deoxynucleoside triphosphate; 400 nM concentrations of primers CIF2 F2, CIF2 R2, MECI P2, MECI P3, RIF5 F10, RIF5 R13, pUB110 R1, and pT181 R1; 800 nM concentrations of primers DCS F2, DCS R2, MECA P4, MECA P7, and IS431 P4; 200 nM concentrations of primers KDP F1, KDP R1, RIF4 F3, and RIF4 R9; 1.25 U of AmpliTaq; and approximately 5 ng of template DNA. PCR amplifications were performed in a DNA Thermal Cycler 480 (Applied Biosystems) with the following parameters: predenaturation for 4 min at 94°C; 30 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 1 min; postextension for 4 min at 72°C; and soaking at 4°C. PCR products (10 µl) were resolved in a 2% SeaKem LE (BioWhittaker Molecular Applications, Rockland, Maine) agarose gel in 0.5x Tris-borate-EDTA buffer (Bio-Rad, Hercules, Calif.) at 100 V and visualized with ethidium bromide.

 

C. Multiplex PCR (Zhang et al. 2005)

All PCR assays were performed directly from bacterial suspensions obtained after the rapid DNA extraction method. An aliquot of 2 µl of this suspension was added to 23 µl of PCR mixture containing 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 2.5 mM MgCl2, 0.2 mM of each deoxynucleoside triphosphate(dATP, dUTP, dGTP, and dCTP) (Invitrogen Inc., Carlsbad, CA), various concentrations of the respective primers (Table 2), and 1.0 unit of Platinum Taq DNA polymerase (Invitrogen Inc., Carlsbad, CA).

The amplification was performed in a GeneAmp PCR system 9700 or 9600 Thermal Cycler (Applied Biosystems, Foster City, CA) beginning with an initial denaturation step at 94°C for 5 min followed by 10 cycles of 94°C for 45 seconds, 65°C for 45 seconds, and 72°C for 1.5 min and another 25 cycles of 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 1.5 min, ending with a final extension step at 72°C for 10 min and followed by a hold at 4°C. For the single target amplification, PCR was run in 23 µl of PCR mixture but containing 0.2 µM of each primer, with cycling parameters beginning with an initial denaturation step at 94°C for 5 min followed by 30 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min, ending with a final extension step at 72°C for 10 min. All PCR assay runs incorporated a reagent control (without template DNA). The PCR amplicons were visualized using a UV light box after electrophoresis on a 2% agarose gel containing 0.5 µg/ml ethidium bromide.

 

D. M-PCR#1 (M-PCR for identification of ccr)(Kondo et al, submitted)

Reaction mixtures contained 10 ng chromosomal DNA, oligonucleotide primers (0.1 mM), 200 mM each dNTPs, Ex Taq buffer, and 2.5 U Ex Taq polymerase (Takara Bio Inc., Kyoto, Japan) in a final volume of 50 ml. The concentration of MgCl2 was 3.2 mM. A Takara PCR Thermal Cycler was used for amplification with an initial denaturation step (94°C, 2 min); 30 cycles of denaturation (94°C, 2 min), annealing (57°C, 1min), extension (72°C, 2 min); and a final elongation at 72°C for 2 min. PCR products were electrophoresed in a 1% agarose-gel with 1 X TAE buffer at 100V for approximately 30 min. The DNA fragments were stained by soaking the agarose gel in 0.01% ethidium bromide solution for 20 min and were visualized using a UV transilluminator.

 

E. For M-PCR#2-5 (M-PCR for identification of mec and J regions) (Kondo et al, submitted)

Reaction mixtures contained 10 ng chromosomal DNA, oligonucleotide primers (0.1 mM), 200 mM each dNTPs, Ex Taq buffer, and 2.5 U Ex Taq polymerase (Takara Bio Inc., Kyoto, Japan) in a final volume of 50 ml. The concentration of MgCl2 was 2.0 mM. A Takara PCR Thermal Cycler was used for amplification with an initial denaturation step (94°C, 2 min); 30 cycles of denaturation (94°C, 2 min), annealing (60°C, 1min), extension (72°C, 2 min); and a final elongation at 72°C for 2 min. PCR products were electrophoresed in a 1% agarose-gel with 1 X TAE buffer at 100V for approximately 30 min. The DNA fragments were stained by soaking the agarose gel in 0.01% ethidium bromide solution for 20 min and were visualized using a UV transilluminator.

 

F. M-PCR#6 (Kondo et al. submitted)

M-PCR#6, we performed long-range PCR using the Expand High Fidelity PCR system following the manufacturer’s recommendations (Roche Diagnostics Co. Indianapolis, IN, USA). Briefly, reactions were performed in a final volume of 50 ml and contained 10 ng template DNA, oligonucleotide primers (0.3 mM), 200 ml of each dNTP, 1x Expand High Fidelity buffer, 1.5 mM MgCl2 , and 2.6 U Expand High Fidelity PCR system enzyme mix. The PCR consisted of denaturation (94°C, 2 min); 10 cycles of denaturation (94°C, 15 s), annealing (50°C, 30 s), and extension (68°C, 8 min); 20 cycles of denaturation (94°C, 15 s), annealing (50°C, 30 s), and extension (68°C, 12 min); and final  elongation (72°C, 7 min).

PCR products were electrophoresed in a 1% agarose-gel with 1 X TAE buffer at 100V for approximately 30 min. The DNA fragments were stained by soaking the agarose gel in 0.01% ethidium bromide solution for 20 min and were visualized using a UV transilluminator.