DNA
   PCR-

2003, Vol. 47, No. 2

Printed in the Republic of Korea

DNA
   PCR-  

  

* ^†^††

 

†
 
 

†† 

(2003. 1. 23 )

Diagnosis of Bovine Theileriosis by Direct PCR and Electrophoresis from Whole Blood Without DNA Extraction

Seong Ho Kang*, Sangmin Jang, Joon-Seok Chae^†, and Yongseong Kim^††

Department of Chemistry, Chonbuk National University, Jeonju 561-756, Korea

†Bio-safety Research Institute, Chonbuk National University, Jeonju 561-756, Korea

††Division of Chemistry and Chemical Engineering, Kyungnam University, Masan 631-701, Korea (Received, January 23, 2003)

.    !" DNA #$ %&
'() *+ ,-./01(PCR) 234 T. buffeli(buffeli/orientalis/sergenti) 16S rRNA 5
6 7 89:; <, 89= DNA>
?@ABC+ DE3 F GB HI3JK. L&5
6 7 89 !" formamide> MN34 'OPQ> N":RCS, TU 1 V> W&? !" XY 01Z[> MN3F FoLT(Formamide Low Temp.) PCRB &N3JK.
' 100-200 nL

> *+ PCR 89( MN3JCS, PCR \](816-bp DNA)Y
?@ABC+ DE3JK. ^ _F T. buffeli( ` a=  'OC+b c= DNA> MN34 dY ef_g h i3JK.

: , , ,-./01,
'

ABSTRACT. We have developed a direct polymerase chain reaction (PCR) and electrophoresis method for the diag- nosis of bovine theileriosis from whole blood DNA analysis without DNA extraction. The technique empolyed a FoLT (formamide, low temp.) technique and was utilized in the diagnosis of bovine theileriosis. Formamide solubilize the blood cells, and the lowered incubation temperatures reduced protein coagulation. 100-200 nL of whole blood and PCR reagents were introduced directly into a PCR tube. After the amplification, the PCR product (816-bp DNA) was intro- duced into the electrophoresis system. The results of this analysis were consist with those obtained using purified DNA.

Keywords: T. buffeli(buffeli/orientalis/sergenti), Direct PCR, Whole Blood, Diagnosis

1. 

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sergenti) 16S rRNA 5
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Table 1. The ratio of PCR reaction mixtures (total volume: 10µL) Sample Blood

(%)

MgCl2

(mM)

10x PCR buffer (%)

dNTP (mM)

Primer(µM) Formamide

(%) Taq³⁾ NW⁴⁾(%) Up¹⁾ Down²⁾

1 2 0 10 0.25 0.4 0.4 16 0.4 50

2 2 0.5 10 0.25 0.4 0.4 16 0.4 48

3 2 1.0 10 0.25 0.4 0.4 16 0.4 46

4 2 1.5 10 0.25 0.4 0.4 16 0.4 44

5 2 2.0 10 0.25 0.4 0.4 16 0.4 442

6 2 2.5 10 0.25 0.4 0.4 16 0.4 40

7 2 3.0 10 0.25 0.4 0.4 16 0.4 38

8 2 3.5 10 0.25 0.4 0.4 16 0.4 36

9 2 4.0 10 0.25 0.4 0.4 16 0.4 34

10 2 4.5 10 0.25 0.4 0.4 16 0.4 32

NW 20 1.5 10 0.25 0.4 0.4 16 0.4 46

NC⁵⁾ 2 1.5 10 0.25 0.4 0.4 16 0.4 44

PC⁶⁾ 2 1.5 10 0.25 0.4 0.4 16 0.4 44

1)Up; forward primer. ²⁾Down: reverse primer. ³⁾Taq: Taq DNA polymerase. ⁴⁾NW: Nuclease free water. ⁵⁾NC: whole blood of non-infected bovine. ⁶⁾PC: pufried DNA from whole blood of infected bovine.

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'

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 7) 89= 816-bp DNA> d  x K. &Ř

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l†0 Ý ³41K. 3® MgCl2 [q æç 2C” ó 816-bp DNA 89& è& l®, r3 Fig. 1. PCR amplification of the 816-bp Theileria buffeli

gene using purified DNA at different MgCl2 concentrations and different cycles of (A) 40, (B) 50 and (C) 70. MgCl2

concentrations: Lane 1, 0 mM; lane 2, 0.5 mM; lane 3, 1.0 mM; lane 4, 1.5 mM; lane 5, 2.0 mM; lane 6, 2.5 mM; lane 7, 3.0 mM; lane 8, 3.5 mM; lane 9, 4.0 mM, and lane 10, 4.5 mM. N: whole blood of non-infected bovine. P: purified DNA from the whole blood of infected bovine. L: 100-bp DNA ladder (100µg/mL).

Table 2. Ratio of PCR reagent at the 0.5 mM, 1.5 mM and 3.0 mM MgCl2 (total volume: 10µL) Sample Blood

(%)

MgCl2

(mM)

10x PCR buffer (%)

dNTP (mM)

Primer(µM) Formamide

(%) Taq³⁾ NW⁴⁾(%) Up¹⁾ Down²⁾

1 2.4 0.5 10 0.25 0.4 0.4 16 0.4 50

2 2.4 1.5 10 0.25 0.4 0.4 16 0.4 44

3 2.4 3.0 10 0.25 0.4 0.4 16 0.4 38

4 1.2 0.5 10 0.25 0.4 0.4 16 0.4 51

5 1.2 1.5 10 0.25 0.4 0.4 16 0.4 45

6 1.2 3.0 10 0.25 0.4 0.4 16 0.4 39

1)Up; forward primer. ²⁾Down: reverse primer. ³⁾Taq: Taq DNA polymerase. ⁴⁾NW: Nuclease free water.

 —F \]& ˜ =K(Fig. 3 lane 6 7).

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DNA> 8934 DE3F ¡, Ë° 'O Þ

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FoLT PCRC+ T. buffeli 16S rRNA 5
6 7 89:; <, 89= 816-bp DNA>
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TU 1V> W&? !" 80^oC &3 XY 01 Z[> MN3JK. PCR( :Ç ,() MgCl² [

Fig. 2. Gel electrophorsis of the 816-bp PCR products (stained with ethidium bromide) from Theileria buffeli under various conditions. Lane 1, 2.4% (v/v) whole blood and 0.5 mM MgCl₂; lane 2, 2.4% (v/v) whole blood and 1.5 mM MgCl2; lane 3, 2.4% (v/v) whole blood and 3.0 mM MgCl2; lane 4, 1.2% (v/v) whole blood and 0.5 mM MgCl₂; lane 5, 1.2% (v/v) whole blood and 1.5 mM MgCl2; lane 6, 1.2% (v/

v) whole blood and 3.0 mM MgCl2; lane 7, 100-bp DNA ladder. PCR cycle=40.

Table 3. Ratio of PCR reagent at the 3.0 mM and 4.5 mM MgCl2 (total volume: 10µL) Sample Blood

(%)

MgCl2

(mM)

10x PCR buffer (%)

dNTP (mM)

Primer(µM) Formamide

(%) Taq³⁾ NW⁴⁾(%) Up¹⁾ Down²⁾

14) - - - -

2 1 0.5 10 0.25 0.4 0.4 16 0.4 39

3 2 1.5 10 0.25 0.4 0.4 16 0.4 38

4 3 3.0 10 0.25 0.4 0.4 16 0.4 37

5 1 0.5 10 0.25 0.4 0.4 16 0.4 33

6 2 1.5 10 0.25 0.4 0.4 16 0.4 32

7 3 3.0 10 0.25 0.4 0.4 16 0.4 31

1)Up; forward primer. ²⁾Down: reverse primer. ³⁾Taq: Taq DNA polymerase. ³⁾NW: Nuclease free water. ⁴⁾100-bp DNA ladder.

Fig. 3. Gel electrophoresis of the 816-bp PCR products from Theileria buffeli under various conditions. Lane 1, 100-bp DNA ladder; lane 2, 1.0% (v/v) whole blood and 3.0 mM MgCl2; lane 3, 2% (v/v) whole blood and 3.0 mM MgCl2; lane 4, 3.0% (v/v) whole blood and 3.0 mM MgCl2; lane 5, 1.0% (v/v) whole blood and 4.5 mM MgCl₂; lane 6, 2.0% (v/

v) whole blood and 4.5 mM MgCl2; and lane 7, 3.0% (v/v) whole blood and 4.5 mM MgCl₂. PCR cycle=40.

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' Þ

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