1

Development of PCR-hybridization for the identification of major Gram

positive bacteria causing bacteremia.

Athicha Mahayotha1, Rosarin Wongvilairat

1, Surang Dejsirilert

2, Tawatchai Sumpradit

1, Anusak Kerdsin

2.

1 Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand.

2 National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Thailand.

E-mail address: aroon59@hotmail.com

Abstract

Blood culture is considered to be a gold standard for diagnosis of bacterial septicemia.

However, this method has some limitations. Molecular techniques for detection of DNA of

bacteria causing septicemia such as PCR and hybridization are sensitive, specific and more

rapid compared with blood culture. The aims of this project are to develop PCR – slot blot

hybridization technique for detection and identification of Gram positive bacteria causing

bacterimia.

16S rRNA gene of gram positive cocci were extracted and amplified by using

universal primer. These fragments of each species were cloned in plasmids and preserved as

clone libraries. Plasmid DNA was amplified by using M13 primer and PCR product used as

DNA template to blot on a nylon membrane. V6 region in 16S rRNA gene of Streptococcus

pneumoniae was amplified by PCR using digoxigenin-11-dUTP. PCR product was an initial

experimental probe to optimize conditions of the hybridization with the reference DNA

blotted on nylon membrane. Various anneal temperature and concentration of SSC buffer

were tested to determine optimum hybridization conditions based on visual observation by

using anti-DIG-AP conjugate, CDP-StarTM.

This study found that the hybridize temperature at 65oC exhibited more specific for

probe of S. aureus, S. epidermidis and E. faecalis and unspecific for Streptococci. At 67oC,

hybridization exhibited more intense signal when used probe of all streptococcal species

without any cross hybridization. The optimal condition of PCR-Slot blot Hybridization that

was chosen as 67oC of the hybridize temperature using for method validation. The lowest of

pure DNA concentration of S. aureus ATCC 25923 which the PCR-Slot blot hybridization

was given as positive results, was 0.7ng/µl. Sensitivity and specificity of PCR-Slot blot

hybridization were highly value (100%). The results revealed that probe design and the

optimized conditions were successful in the detection of gram positive septicemia-bacteria.

Key words: Septicemia, Cultural method, PCR, Hybridization, 16S rRNA gene.

2

Introduction

Septicemia is the presence of bacteria in blood ( bacteremia ) and is often

associated with severe disease. This disease is a serious; life- threatening infection that gets

worse very quickly. In case of severe sepsis is a serious condition that requires a hospital

stay and an intensive care unit (ICU) for admission (Amersfoort et al., 2003; Cunha, 2003;

Abraham, 1999; Ammerlaan et al., 2009). Early diagnosis of this disease is a key for

prevention of septic shock progression and associated with the correctly treatment of

clinicians. Physical examination and laboratory confirmation are used as a tool in septicemia

diagnosis. Especially laboratory tests play an important role in identifying the infectious

agent causing the infections (Agnihotri, 2004). Blood culture is considered to be the gold

standard for diagnosing bacterial septicemia (Baron et al., 2005; Chen et al., 2008). However,

this method has some limitations, including; a long time for growth of an organism(48 – 72

hrs), unacceptably low sensitivities in many clinical situations(40%) and inability to grow

by culturing or non-cultural bacteria (Bone, 1994; Keen et al., 2002; Thompson & Madeo,

1994; Craven, 2004 ). Molecular detection of septicemia bacteria based on DNA-based

detection methods including PCR and hybridization, both of which are sensitive and specific

and are rapid compared with blood culture (Kunakorn et al., 2000; Fu et al., 2002; Kim et al.,

2006; Hony et al., 2003; Mako et al., 2002). Here, we design one primer and one probe using

PCR-slot blot hybridization to recognize different strains within gram positive septicemia -

bacteria and will provide an rapid method for identification gram positive bacteria which

frequently in septicemia. And this should be applied in the hospital routine diagnostic

laboratory for early diagnosis gram positive bacteria.

Materials and Methods

Bacterial strains and culture. 11Reference septicemia bacteria (Table 1) were obtained from

the Culture Collection at the National Institute of Health, Department of Medical Sciences,

Ministry of Public Health (NIH, Nontaburi, Thailand). All strains were cultured on agars and

incubated 1-3 days depend on strains and species. Pure colonies are selected and sub-cultured

again for DNA extraction.

Extraction of bacterial DNA and DNA amplification. Bacterial cells of each strains were

harvested in sterile DW. DNA were extracted by using High pure PCR Template preparation

kit (Roche Diagnostic, USA) according to the manufacturer’s protocol. PCR amplification

of the 16S rRNA regions were conducted by PCR Thermal cycler (TC320, ASTEC, Japan)

using forward and reverse universal bacterial primers; 27F and 1492R (Table 2). PCR

reactions were performed in the final volume of 25 μl containing: 2.5 μl of 10X PCR reaction

buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 20 mM MgCl2 and enhancer solution), 0.5 μl

of 10 mM dNTP mixtures, 1.25 μl of each of 10 pmol 27F and 1492R, 0.2 μl of 5 unit/μl of

Taq DNA polymerase (i-Taq), 1.0 μl of DNA template solution, and 18.3 μl of double

sterile distilled water.

3 Table 1: Reference strains of gram positive septicemia bacteria.

Bacterial species

Staphylococcus aureus ATCC 25923 DMST 8840,

S. epidermidis ATCC 12228 DMST 15505,

Sterptococus pyogenes DMST 17020,

S. agalactiae DMST 17129,

S. equi ATCC 33398T DMST 18774,

S. mitis DMST 8775,

S. bovis ATCC 33317T DMST 18769,

S. dysgalactiae DMST 173000,

S. pnuemoniae ATCC 49619 DMST 7945,

S. mutans ATCC 25175T DMST18777 Enterococcus faecalis ATCC 29212 DMST 4736

The PCR conditions were operated by pre-denaturation at 95oC for 3 min, followed by

35 cycles of denaturation at 95oC for 1 min, annealing at 58

oC for 45 sec, and extension at

72oC for 1 min, and final extension at 72

oC for 7 min. The PCR products were analyzed by 1

% agarose gel electrophoresis, and purified with Nucleospin®

Extract II (PCR Clean-up) Kit,

according to the manufacturer’s protocol.

Table 2: Sequences of primers

Name Sequences 5’- 3’ Expected size of product Universal primers:

primer 27F AGA GTT TGA TCM TGG CTC AG 1331 bp

primer 1492R GGT TAC CTT GTT ACG ACT T

vector primers :

M13F TGT AAA ACG ACG GCC AGT 1446 bp

M13R CAG GAA ACA GCT ATG ACC

V6 Primer

V6-F (961-978)* TCG ATG CAA CGC GAA GAA 125 bp

V6-R (1062-1085) ACA TTT CAC AAC ACG AGC TGA CGA

* = 16S ribosomal RNA gene position of E. coli (Chadravorty et al., 2007)

16S ribosomal RNA gene cloning. Purified 16S rDNA PCR products were cloned by using

Strataclone PCR Cloning kit (Stratagene, USA). In ligation reaction composed of 3.5 μl of

purified 16S fragments, 3.0 μl of ligation buffer and 1.0 μl of Strataclone vector. Then, the

mixtures were incubated at room temperature for 7 min after that kept the mixture on ice.

Transformation reactions were prepared, by adding 2.0 μl of ligation mixture to E. coli cells

(competent cells), incubated on ice for 20 min.

Transformations were performed at 42oC, 45 second and kept them on ice. 200 μl of

LB broth were added in competent cells and incubated at 37oC for 1 hr. After incubation, the

E. coli cells were spread on LB agar with X-gal and ampicillin, incubated at 37°C, overnight.

4 White colonies were selected to propagation of recombinant plasmid by streak on LB agar

with X-gal and ampicillin. Plasmid extractions were performed by using Plasmid DNA

Purification kit (Nucleospin®, Germany).

The recombinant plasmids were tested for inserted fragment by PCR using M13

primer. PCR reactions were performed in the final volume of 25 μl containing: 2.5 μl of 10X

PCR reaction buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 20 mM MgCl2 and enhancer

solution), 0.5 μl of 10 mM dNTP mixtures, 1.25 μl of each of 10 pmol M13F and M13R, 0.2

μl of 5 unit/μl of Taq DNA polymerase (i-Taq), 1.0 μl of DNA template solution, and 18.3

μl of double sterile distilled water. The PCR conditions were operated similar in PCR

amplification of the 16S rRNA regions. The PCR products were analyzed by 1% agarose gel

electrophoresis. Each recombinant clones of all reference bacteria were stored at -70oC as

DNA templates to be applied hybridization.

Preparation of reference 16S rRNA gene for blotting on membrane. Each reference

bacterial genomic 16S rRNA gene from recombinant clones were amplified by using M13

primers. PCR reactions were performed in the final volume of 50 μl containing: 5.0 μl of 10X

PCR reaction buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 20 mM MgCl2), 1.0 μl of 10

mM dNTP mixtures, 2.5 μl of each of 10 pmol of M13 forward and reverse primers, 0.4 μl of

5 unit/μl of Taq DNA polymerase (i-Taq), 2.0 μl of DNA template solution, and 36.6 μl of

double sterile distilled water. The PCR conditions were operated similar in PCR

amplification of the 16S rRNA regions. DNA concentrations were determined by UV-

Spectrophotometer. 50 ng/ml of each target DNA was prepared and boiled at 100oC for 10

min before apply on nylon membrane by slot blot machine (Bio-RAD, USA). Then

membranes were fixed by UV-Transluminator for 30 sec and were stored at -20oC.

Probe labeling. This study, probe was the PCR product of V6 of 16S rRNA gene from

bacteria (Chadravorty et al., 2007). PCR reaction was performed in a final volume of 50 μl

containing: 5.0 μl of 10X PCR reaction buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 20

mM MgCl2 , 5.0 μl of 10 mM dNTP mixtures with digoxigenin-11-dUTP, 2.5 μl of each of

10 pmol of forward and reverse primer for V6 region of 16S rRNA, 0.4 μl of 5 unit/μl of Taq

DNA polymerase (i-Taq), 2.0 μl of DNA template solution, and 32.6 μl of double sterile

distilled water. The PCR condition was operated by pre-denaturation at 95oC for 3 min,

followed by 35 cycles of denaturation at 95oC for 1 min, annealing at 58

oC for 30 sec, and

extension at 72oC for 1 min, and final extension at 72

oC for 7 min. Probe concentration was

determined by UV-Spectrophotometer.

Probe hybridization setting and optimized method. The nylon membrane was placed on

filter paper and let it to room temperature. After membrane was cut and labeled, inserted it to

rolling bottle and added 5 ml of DIG Easy Hybridization (Roche Diagnostic, USA). Pre-

hybridize by placing rolling bottle rotated at 65oC for 1 hour in hybridization incubator. The

specific probe of S. pneumoniae ATCC 49619 was an initial experimental probe to optimize

condition of the hybridization. 2 μl of 50 ng/ml of labeled probe was added in 500 μl of DIG

Easy Hybridization and denatured at 100oC for 2 min. Add 2 μl of denatured probe to rolling

bottle, the hybridization temperature will be variable from 58 - 70oC, overnight.

5

Washing of the membrane by variable concentration of standard saline-citrate buffer,

SSC (Roche Diagnostic, USA) at 68oC for 15 min from 2X, 0.5X and 0.1X, respectively.

Membrane was soaked in plastic box with maleic acid buffer and drained. After that added 40

ml of blocking reagent to membrane, mixed gently and shake at room temperature for 1 hour.

Prepared conjugate by mixing of 2.5 μl of Anti-DIG-AP conjugate and 10 ml of blocking

reagent gently and poured to membrane in plastic box, shake gently at room temperature for

30 min. Membrane washing by wash buffer at room temperature for 20 min, 3 times and

maleic acid buffer for 5 min, twice. In the visualization step, rinsed membrane briefly in 20

ml of detection buffer. Placed membrane on plastic sheet and applied 150 μl of 1: 100 diluted

of CDP-StarTM

(Roche Diagnostic, USA). Immediately covered the membrane with the

second sheet to spread the substrate evenly and without air bubbles over the membrane,

incubate for 1 min. Exposed to X-ray film for 30 sec, placed X-ray film in development

buffer for 3 min. Stop reaction on X-ray film by soaking film in water for 1 min and then film

was fixed in fixing buffer for 5 min and dried.

The testing of the optimized conditions of hybridization. The other probes of gram

positive bacteria were tested with these conditions, including S. aureus, S. epidermidis, S.

pyogenes, S. agalactiae, S. equi, S. mitis, S. bovis, S. dysgalactiae, S. mutans and E. faecalis.

Method validation

Method validation: limit of detection, sensitivity, specificity are determined by using

positive blood culture in clinical specimen under the optimal PCR-Slot blot Hybridization

conditions in comparison with conventional blood culture results (Alonzo & Pepe 1999).

Limit of Detection. The detection limit of PCR-Slot blot Hybridization had been

determined by using pure DNA 12- diluted series of S. aureus ATCC 25923. DNA

concentrations were detected and had been started with 43.6, 22.8, 7.7, 0.7 ng/µl and below

limit of detection (not detectable).

Sensitivity and specificity of the PCR-Slot blot hybridization. The sensitivity and

specificity of the PCR-Slot blot hybridization was evaluated by testing with 13 blood culture

samples that had positive results within Gram positive septicemia bacteria such as S. aureus,

E. faecalis, S. pneumonia, S. mutans and S. agalactiae and 30 blood culture samples that had

negative results within Gram positive septicemia bacteria. The other groups of blood culture

samples that had positive results in the other gram positive bacteria, included 12 samples of

coagulase-negative Staphylococci, 2 samples of E. faecium, a sample of S. suis and 5 samples

of Bacillus spp.) were also determined its sensitivity and specificity with this method.

Results

In the step of DNA template preparation comprised of reference bacterial cultures,

extraction of bacterial nucleic acids, DNA target amplification by using universal primer and

DNA target cloning. 1,446 bps- DNA target-template of all gram bacteria blotted on

membrane were synthesized by using M13 primers via PCR, shown in Fig. 1.

6

Polynucleotide probe-labeled sized 125 bps was synthesized by using a primer pair (V6F,

V6R) and DIG-11-dUTPs via PCR, shown in Fig. 2.

The result of initial experimental probe to optimize conditions of the hybridization,

showed that all gram positive bacteria were positive under the hybridized temperatures varied

from 58 to 62oC and were negative at hybridized temperature at 70

oC (Fig. 3). Interesting,

only S. pneumoniae was discriminated from other gram positive bacteria at the optimized

temperatures varied from 65 to 68oC (Fig. 3).

Fig. 2: Polynucleotide probe-labeled sized 125 bps was synthesized by using a primer pair (V6F, V6R) and DIG-11-dUTPs via PCR.

A B

Fig. 1: A = PCR products from 16S rRNA gene amplification of reference bacteria by universal primer (27F, 1492R), B = PCR products from plasmid DNA amplification by using M13 primer as DNA template and blotted on a nylon membrane.

C: Cross hybridization D: Specific hybridization E: Specific hybridization F: No hybridization

(light signal)

Fig. 3: C=Cross hybridization of S. pneumoniae probe with gram positive septicemia target bacteria at

temperature hybridization 58 oC , D = Specific hybridization of S. pneumoniae probe with S. pneumoniae

target bacteria at temperature hybridization 65 oC, E = Specific hybridization of S. pneumoniae probe with

S. pneumoniae target bacteria at temperature hybridization 68oC , and F = No hybridization of S.

pneumonia probe with gram positive septicemia target bacteria temperature hybridization 70 oC.

7

The hybridize temperature varied from 65 to 67oC was tested again with the other probes

of gram positive bacteria included S. pyogenes, S. agalactiae, S. equi, S. mitis, S. bovis, S.

dysgalactiae, S. mutans, S. aureus, S. epidermidis and E. faecalis . These results showed that

all gram positive bactertia were positive at hybridized temperature at 67oC (Table 3).

Table 3: Cross hybridization and Specific hybridization of gram positive septicemia-bacteria

probes with gram positive septicemia target bacteria at temperature hybridization

65oC and 67

oC.

Specific probes Temperature of Hybridization

65oC 67

oC

S. pyogenes Cross hybridization Specific hybridization

S. agalactiae Cross hybridization Specific hybridization

S. equi Cross hybridization Specific hybridization

S. mitis Cross hybridization Specific hybridization

S. bovis Cross hybridization Specific hybridization

S. dysgalactiae Cross hybridization Specific hybridization

S. mutans Cross hybridization Specific hybridization

S. aureus Specific hybridization Specific hybridization

S. epidermidis Specific hybridization Specific hybridization

E. faecalis Specific hybridization Specific hybridization

(light signal)

Method validation

The optimal hybridization temperature for the PCR-Slot blot Hybridization was at

67oC, positive blood culture in clinical specimens are tested by this technique to determine

method validation (Alonzo & Pepe 1999): limit of detection, sensitivity, specificity in

comparison with conventional blood culture results.

The limit of detection, pure DNA concentration of S. aureus ATCC 25923 which the

PCR-Slot blot Hybridization was detected as positive results, was 0.7 ng/µl. In other hand,

this method had been detected as negative results in the non-detectable DNA samples.

In the group of positive septicemia bacteria were found that all of 13 samples

(sensitive, 100%) were detected as positive results by the PCR-Slot blot hybridization. And

all of 30 negative samples (specific, 100%) were tested as negative results by this method

also. For the other groups of gram positive bacteria, Coagulase-negative Staphylococci, there

were 5 of 12 samples that reacted hybridization with S. aureus, 3 of 12 samples had reacted

hybridization with S. epidermidis and other samples had been negative results. For positive

sample of S. suis when was tested with this technique, found that had cross hybridization with

S. dysgalactiae. In the last group, positive samples of E. faecium and Bacillus spp., all of 7

samples did not show positive hybridization with these septicemia bacteria as a supplement to

negative results.

8 Discussion

The results of this study showed that gram positive bacteria causing frequently

septicemia were positive under the hybridize temperature varied from 58 to 62oC and were

negative at hybridize temperature at 70oC. Interesting, only S. pneumoniae was discriminated

from other gram positive septicemia-bacteria at the optimized temperature varied from 65 to

68oC. But the detection signal of hybridization at 68

oC was light signal as a consequence that

this temperature was higher than the melting temperature (Table 4) and may have substantial

effects on weaker hydrogen bonding between probe-target sequences (DIG Application

Manual, Roche applied science, 2009). This polynucleotide probe will benefit for the

identification S. pneumoniae in heamo-cultures samples by using the specific optimized

conditions (Davis & Fuller 1991; Zhang et al., 1995; Chadravorty et al., 2007).

The hybridize temperature varied from 65 to 67oC was tested again with the other

specific probes of gram positive septicemia-bacteria included S. pyogenes, S. agalactiae, S.

equi, S. mitis, S. bovis, S. dysgalactiae, S. mutans, S. aureus, S. epidermidis and E. faecalis.

The hybridize temperature at 65oC exhibited more specific for S. aureus, S. epidermidis and

E. faecalis and unspecific for all Streptococcus spp. From the similarity matrix of multiple

sequence alignments of V6 region (961-1085) including S. aureus strain ATCC 14458

(GenBank: DQ269498), S. epidermidis strain ATCC 12228 (NC_004461), E. faecalis strain

ATCC 29212 (GenBank: GU585587), S. pneumonia strain ATCC 49619 (GenBank:

AY281082) and S. dysgalactiae subsp. equisimilis ATCC 12394 (NC_017567), found that

differentiation of Streptococci from Enterococci and Staphylococci varying between 15- 30%

in comparative sequence alignments and direct effect on specific hybridization with

Enterococci and Staphylococci (Fig. 4).

gi|S.aureus TCGAAGCAAC GCGAAGAACC TTACCAAATC TTGACATCCT T–TGACAACT CTAGAGATAG gi|S.epidermidis TCGAAGCAAC GCGAAGAACC TTACCAAATC TTGACATCCT C- TGACCCCT CTAGAGATAG gi|E.faecalis TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCT T- TGACCACT CTAGAGATAG gi|S.pneumoniae TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCC TCTGACCGCT CTAGAGATAG gi|S.dysgalactiae TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCT CCTGACCGGT CTAGAGATAG

********** ********** ****** ** ********* **** * ********** gi|S.aureus AGCCTTCCCC TTCGGGGGAC AAAGTGACAG GTGGTGCATG GTTGTCGTCA GCTCGTGTCG gi|S.epidermidis AGTTTTCCCC TTCGGGGGAC AGAGTGACAG GTGGTGCATG GTTGTCGTCA GCTCGTGTCG gi|E.faecalis AGCTTTCCCT TC—GGGGAC AAAGTGACAG GTGGTGCATG GTTGTCGTCA GCTCGTGTCG gi|S.pneumoniae AGTTTTCCTT C ---GGGACA GAGGTGACAG GTGGTGCATG GTTGTCGTCA GCTCGTGTCG

gi|S.dysgalactiae GCTTTCCCTT C ---GGGGCA GGAGTGACAG GTGGTGCATG GTTGTCGTCA GCTCGTGTCG

* ** *** ******* ********** ********** ********** gi|S.aureus TGAGATGT gi|S.epidermidis TGAGATGT gi|E.faecalis TGAGATGT gi|S.pneumoniae TGAGATGT gi|S.dysgalactiae TGAGATGT

********

Fig. 4: Similarity matrix of multiple sequence alignments of V6 region sequences (961-1085) among S. aureus strain ATCC 14458, S. epidermidis strain ATCC 12228, E. faecalis strain ATCC 29212, S. pneumonia strain ATCC 49619 and S. dysgalactiae subsp. equisimilis ATCC 12394.

9 Table 4: The melting temperature (Tm) of V6- Probes which were calculated from reference

bacterial strains.

V6 - Probe type

(961-1085)*

length

(bp) %GC

Tm (basic)

°C ** Tm(DIG Esay)

°C***

Thyb(DIG Esay)

°C Reference Sequence

E. faecalis

strain ATCC29212

S. aureus

strain ATCC14458

S. epidermidis

strain ATCC12228

S. pneumonia

strain ATCC49619

S. dysgalactiae subsp.

equisimilis ATCC12394

S. pyogenes

strain ATCC12344

S. mutans

strain ATCC25175

S. equi subsp.

zooepidemicus

ATCC 35246

S. bovis

strain ATCC 33317

S. agalactiae

strain ATCC 13813

S. mitis

strain ATCC 49456

125

127

127

125

125

125

125

125

125

125

125

50

49

50

51

54

50

48

46

49

51

51

80

80

80

81

81

80

79

79

80

81

81

65

65

66

66

67

66

65

64

65

66

66

40-45

40-45

41-46

41-46

42-48

41-46

40-45

39- 44

40-45

41-46

41-46

GU585587

DQ269498

NC_004461

AY281082

NC_017567

AB002521

DQ303189

NC_017582

AB002482

NR_040821

AF003929

* = 16S ribosomal RNA gene position of E. coli (Chadravorty et al., 2007)

** = BioMath Calculators, Tm Calculations for Oligos

*** = The optimum hybridization temperature (Thyb) in DIG Easy Hyb buffer

Tm = 49.82 + 0.41 (% G + C) - 600/L

Thyb = Tm – (20° to 25°C)

Within Streptococci group, the results of V6 region sequences alignments had shown

the aligned score between 80-100 % similar sequences (Fig. 5). When hybridize temperature

was increased to 67oC, each specific probe hybridized to each specific Streptococci within

Streptococci group with intense signal without any cross hybridization. The hybridized

temperature at 67 oC, results in probe specific hybridize with S. pneumonia strain ATCC

49619 and S. mitis strain ATCC 49456, eventhough both of them were 100% similarity and

51% GC content (Fig.5). The finding indicated that there were many factors that influenced

on specificity of probe hybridization such as hybridize temperature, the differentiation of

sequence, % GC contents, sequence length, hybrid buffer, also probe concentration (Brown

1993; Greisen et al., 1994; Heuer et al., 1999; Clarridge 2004).

The optimal condition of PCR-Slot blot Hybridization that was chosen as 67oC of the

hybridize temperature using for method validation due to this condition could classified all

reference strains of gram positive septicemia-bacteria by using only one primer pair for probe

synthesis (Greisen et al., 1994; Rodríguez et al., 1996). The lowest of pure DNA

concentration of S. aureus ATCC 25923 which the PCR-Slot blot Hybridization was given as

positive results, was 0.7ng/µl. Sensitivity and specificity of PCR-Slot blot hybridization were

10 highly value, 100% (Alonzo & Pepe 1999); Zhang et al., 1995; Borchardt et al., 2004). In the

cases of Coagulase-negative Staphylococci were not confirmed to bacterial species, these

cases may be identified to many species of Staphylococci. Few samples hybridized with S.

epidermidis which may truly be S. epidermidis, a more detailed identification test could be

experimental to confirm this hybridization by using biochemical tests as in analytical profile

index tests methods. For some samples of cross hybridization with S. aureus are therefore

critically important in false reports in some species. A false negative may be perceived as the

samples are not allowed time to cool for 30 minutes at room temperature or 10 minutes in the

freezer, given the fact that the serum will be sufficient for melting (Sperber & Tatini, 1975;

Essers and Radebold, 1980; Dickson & Marples, 1986). Cross hybridization between S. suis

and S. dysgalactiae revealed the closest similarity between theV6-16SrRNA region of these

two bacteria, increasing the hybridize temperature would be increase the discrimination

power in order to separate S. suis and S. dysgalactiae (Fig.6). For the last group, E. faecium

and Bacillus spp., all of 7 samples, results found that there were no cross hybridization with

these septicemia bacteria that supporting the negative results.

gi|S.dysgalactiae TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCT CCTGACCGGT CTAGAGATAG gi|S.agalactiae TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCT TCTGACCGGC CTAGAGATAG gi|S.pneumoniae TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCC TCTGACCGCT CTAGAGATAG gi|S.mitis TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCC TCTGACCGCT CTAGAGATAG gi|S.pyogenes TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCC GATGCCCGCT CTAGAGATAG gi|S.mutans TCGATGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCC GATGCTATTC TTAGAGATAG gi|S.equi TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCC GATGCTATTC TTAGAGATAA gi|S.bovis TCGATGCAAC CGCAAGAACC TTACCAGGTC TTGACATCCC GATGCTATTC TTAGAGATAG

**** ***** ********* ********** ********* ** *********

gi|S.dysgalactiae GCTTTCCCTT CGGGGCAGGA GTGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTCGTGA gi|S.agalactiae GCTTTCTCTT CGGAGCAGAA GTGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTCGTGA gi|S.pneumoniae AGTTTTCCTT CGGGACAGAG GTGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTCGTGA gi|S.mitis AGTTTTCCTT CGGGACAGAG GTGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTCGTGA gi|S.pyogenes AGTTTTACTT CGGTACATCG GTGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTCGTGA gi|S.mutans GAAGTTACTT CGGTACATCG GAGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTCGTGA gi|S.equi GAAGTTACTT CGGTACATTG GAGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTCGTGA gi|S.bovis GGTTTCTCTT CGGAACATCG GTGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTTGTGA

* *** *** ** * ******** ********** ********** ***** **** gi|S.dysgalactiae GATGT gi|S.agalactiae GATGT gi|S.pneumoniae GATGT gi|S.mitis GATGT gi|S.pyogenes GATGT gi|S.mutans GATGT gi|S.equi GATGT gi|S.bovis GATGT

*****

Fig. 5: Similarity matrix of multiple sequence alignments of V6 region sequences (961-1085) within Streptococci composed S. pneumonia strain ATCC 49619, S. dysgalactiae subsp. equisimilis ATCC 12394, S. pyogenes strain ATCC 12344, S. mutans strain ATCC 25175, S. equi subsp. zooepidemicus ATCC 35246, S. bovis strain ATCC 33317, S. agalactiae strain ATCC 13813 and S. mitis strain ATCC 49456.

11

Conclusion

The results revealed that probe design and the optimized conditions were successful in

the detection of gram positive septicemia-bacteria. From these results, polynucleotide probes

targeting a region of the V6 of 16S rRNA gene probe, and the specific optimized

hybridization conditions at 67oC proved to be more specific for the identification each

septicemia- gram positive bacteria. This finding is a major progress; however further

investigation of the gram negative septicemia-bacteria would be required for a broader

identification of septicemia bacteria.

Acknowledgments

We would like to thank you the department of Medical Sciences, Ministry of Public

Health, Thailand to support the finance of this project and really thank you the faculty of

Medical Science, Naresuan University to give us about introduction, equipment for all of the

project processes. And thank you of officer of the Khon Kaen hospital and Regional Medical

Science Center, Khon Kaen to help me to collect blood culture samples and sample

preparation.

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gi|S.dysgalactiae TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCT CCTGACCGGT CTAGAGATAG gi|S.suis TCGATGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCC GATGACCGCC CTAGAGATAG

**** ***** ********** ********** ********* ****** ********** gi|S.dysgalactiae GCTTTCCCTT CGGGGCAGGA GTGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTCGTGA gi|S.suis GGTTTCTCTT CGGAGCATCG GTGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTCGTGA

* **** *** *** *** ********** ********** ********** ********** gi|S.dysgalactiae GATGT gi|S.suis GATGT

*****

Fig. 6: Similarity matrix created of sequence alignments of V6 region sequences (961-1085) between S. dysgalactiae subsp. equisimilis ATCC 12394 and S. suis strain ATCC 43765.

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