ISH uses oligonucleotide probes to

ISH uses oligonucleotide probes to detect complementary
nucleic acids sequences. This method exploits
the ability of nucleic acids to anneal to one another in a
very specific complementary way to form hybrids. The
probes are specific because they are built from, and are
complementary to, selected nucleic acids sequences
which are unique to a given microorganism, species or
group. The probes can target either DNA or RNA
molecules. Use of rRNA sequences (5S, 16S and 23S)
to study phylogenetic relationships on the basis of their
divergence and to develop determinative hybridization
probes is now well established (Amann et al., 1995).
The sequencing of more than 2500 of the bacterial
species 16S rRNA currently confers on these genes a
very high informative value at a phylogenetic level.
Sequence comparisons make it possible to define
targeted regions which are perfectly conserved within
different taxonomic levels and consequently specific
to these levels, from domains to subspecies. Also of
interest in targeting rRNA molecules is the high level
of rRNA molecules copies, which is in turn linked to
the large number of ribosomes per cell. The number of
ribosomes varies, generally between 103 and 105 per
bacteria, according to the species and to the physiological
state of the cells, and is directly correlated with
the cellular growth rate (Amann et al., 1995). Probes specific to rRNA (mainly 16S and 23S rRNA sequences)
have become the standard tools for organism
identification (Olsen et al., 1986).
Several oligonucleotide probes are commercially
available and the choice of whether to use them or
design new, specific ones depends largely on the
application. To find the continuous target sequence
unique to a specific microorganism, researchers rely
on computer-aided sequence comparison available in
the Ribosomal Database Project (RDP) (Maidak et al.,
1996), in GenBank for DNA (Benson et al., 1999), or
in the ARB software package (Max Planck Institute,
Bremen, Germany). For better specificity, the target
sequence should be short (15 to 30 bases) and have at
least two to three different nucleotides with homologous
sequences of closely related organisms (DeLong,
1993). Early work on in situ hybridization relied on
radioactive probes to reveal and detect the probetarget
hybrid (Olsen et al., 1986). Current work on
rRNA in situ hybridization uses fluorescent-labeled
nucleotide probes almost exclusively to detect hybridization
(FISH). The popularity of the FISH technique
is due to its advantages over radioactive labeling,
which include sensitivity, speed of visualization of
single cells (by means of microscopy or cytometrical
devices), stability of the hybridization products,
safety, diminished detection time, multiple labels
(multiple colors) and ease of use (Richardson et al.,
1991; DeLong, 1993; Swinger and Tucker, 1996).
Fluorescein and rhodamine dyes are the most frequently
used fluorochromes (DeLong et al., 1989;
Amann et al., 1990; Manz et al., 1992; 1993; Wagner
et al., 1994), but the CY3 dye, brighter and leading to
lower non-specific fluorescence than the other fluorochromes,
has recently been attracting more attention
(Wessendorf and Brelje, 1992; Glockner et al., 1996;
Zarda et al., 1997; Kalmbach et al., 1997a; Ouverney
and Fuhrman, 1999). In practice, FISH procedure
steps are: cell fixation, hybridization (specificity and
stringency depend on hybridization temperature and
time, salt concentration, probe concentration and
length), post-hybridization washing (to remove
unbound or non-specifically bound material) and
detection. Hybridized cells are usually detected by
epifluorescence microscopy and a counterstain such
as DAPI (40,60-diamidino-2-phenylindole) or orange
acridine is used to determine the total number of cells.
Depending on the concentration of targeted cells in
the sample and to increase resolution, FISH detection
can be performed by means of flow or solid-phase
cytometry. Flow cytometry enables quantification of
the fluorescence intensities for each target-probe
hybrid (Fuchs et al., 1998).
For TC, the development of a specific 16S rRNA
probe for this group is not possible, since the coliform
group as defined by the water industry is a group
containing bacteria from genera that are phylogenetically
different. As a result, probes (Table 3) were
developed for the Enterobacteriaceae family rather
than for TC. The first, ENTERO (Mittelman et al.,
1997) was developed for clinical detection of urinary
tract infections. The second, ENT1 (Loge et al., 1999)
was developed for application on wastewater samples.
These two probes are different in their composition
and in the hybridization conditions. The ENTERO
probe is longer-25 bases with a C +G content of 48%
compared to 17 bases and a 70% C +G for the ENT1
probe. Research within the RDP (Maidak et al., 1996)
showed higher specificity for the ENT1 probe for
Enterobacteriaceae species than the ENTERO probe
(Rompre´ and Baudart, NSERC Industrial Chair on
Drinking Water, Ecole Polytechnique Montreal, personal
communication).
Sequences of rRNA target probes for the detection
of E. coli have also been published. The EC1531
probe (Poulsen et al., 1994), complementary to a 23S
rRNA sequence, is composed of 20 nucleotides with a
C +G content of 55%. Shi et al. (1999) used this
probe for the detection of E. coli after modifying the
hybridization conditions, but no results were mentioned.
The probe had only been used as a control in
an experiment using a microcosm extract spiked with
E. coli DNA. More recently, Regnault et al. (2000)
developed the Colinsitu probe for the detection of E.
coli and E. fergusonii in urine, water (rivers and
sewage) and food samples. The Colinsitu probe complements
a 16S rRNA sequence and is composed of
24 nucleotides (C +G content of 46%). This probe
showed a good specificity for the visualization of E.
coli from the various media tested and has newly been
used with success for the detection of E. coli in
drinking water samples (Delabre et al., 2001).
The FISH technique appears to be a highly specific
detection method at a cellular level; however, it may
have some limitations when applied to the detection of
nutrient starved bacterial cells disseminated in drink-ing waters. These limitations are linked to a low
bacterial ribosome content and thus to the small
number of 16S rRNA targets of these cells, which
induces weak fluorescent hybridization signals
(Amann et al., 1995; Lebaron et al., 1997). This is
probably why FISH is rarely described in the drinking
water literature at this time. As a consequence, fluorescence
amplification systems, such as multiple
probing (instead of monolabeled probing in the conventional
FISH technique), interactive properties of
the biotine–avidine complex and/or horseradish peroxydase
conjugated with fluorochrome–tyramidesubstratum,
could represent a way to increase the
intensity of the fluorescent signal given off by starved
hybridized cells (Lebaron et al., 1997; Scho¨nhuber et
al., 1997). Recently, Prescott and Fricker (1999)
reported the use of a peptide nucleic acid (PNA)
probe for in situ hybridization and detection of E.
coli in tap water. The hybridization results were
similar to those yielded by the plate count method.
A PNA is a synthetic nucleic acid in which the sugar
backbone is replaced with a peptide backbone. The
advantages to using PNA instead of DNA probes
include better resistance to nuclease attack, hybridization
independent of salt concentration, more specific
binding and shorter probes sequences, to achieve
greater sensitivity. Since the PNA probe binds more
strongly to the target site, hybridization reaction can
be completed in a shorter time (30 min). To our
knowledge, except for the E. coli PNA probe (Prescott
and Fricker, 1999), none of these probes has been
used for coliforms or pathogenic bacteria monitoring
in drinking water. Before they are accepted as a
sensitive coliform enumeration method, they must
first be subjected to a validation stage so that the
probe most suitable for the samples analyzed can be
chosen and to optimize the hybridization conditions if
necessary.
Cell viability is a major question arising in the
utilization of FISH for monitoring contamination of specificity of the oligonucleotidic probe and on the
stringency of hybridization conditions used. Identification
of the target sequence remains tedious, however,
and FISH cannot be applied to the detection of nonphylogenetically
identified micro-organisms, such as
coliforms. In this case, work can be done on Enterobacteriaceae,
the nearest phylogenetically identified
group that can be considered as a potential indicator of
fecal contamination.
drinking water. It is established that the rRNA content
of the bacterial cells is correlated directly with the
growth rate (Amann et al., 1995). However, the rRNA
content of a bacteria may not completely reflect its
physiological status. It appears that a small number of
rRNA molecules can remain for a relatively long
period after the loss of culturability. S. aureus and E.
coli rRNA were still detected 48 h after moderate heat
activation or UV irradiation (McKillip et al., 1998).
Sheridan et al. (1998) still found E. coli 16S rRNA 16
h after thermal inactivation at 100, 80 and 60 C.
Finally, Prescott and Fricker (1999) exposed an E. coli
strain to 1.5 mg/l of chlorine for up to 30 min. Using
the PNA probe and the tyramide fluorescent amplification
system (TSA kit; Perkin Elmer Life Science,
Ontario, Canada), they still detected bacteria immediately
after chlorination, while no cells were detected by
the plate counts or Colilert methods. Two weeks after
chlorination, 10% of the cells were still being detected
by the PNA probe-TSA kit. This can be viewed as an
advantage to the method in that non-culturable coliforms
can be detected, or as a disadvantage in that dead
cells might be enumerated. Further investigations on
the physiological state of the bacteria enumerated by

ผลลัพธ์ (ไทย) 1:

ISH uses oligonucleotide probes to detect complementarynucleic acids sequences. This method exploitsthe ability of nucleic acids to anneal to one another in avery specific complementary way to form hybrids. Theprobes are specific because they are built from, and arecomplementary to, selected nucleic acids sequenceswhich are unique to a given microorganism, species orgroup. The probes can target either DNA or RNAmolecules. Use of rRNA sequences (5S, 16S and 23S)to study phylogenetic relationships on the basis of theirdivergence and to develop determinative hybridizationprobes is now well established (Amann et al., 1995).The sequencing of more than 2500 of the bacterialspecies 16S rRNA currently confers on these genes avery high informative value at a phylogenetic level.Sequence comparisons make it possible to definetargeted regions which are perfectly conserved withindifferent taxonomic levels and consequently specificto these levels, from domains to subspecies. Also ofinterest in targeting rRNA molecules is the high levelof rRNA molecules copies, which is in turn linked tothe large number of ribosomes per cell. The number ofribosomes varies, generally between 103 and 105 perbacteria, according to the species and to the physiologicalstate of the cells, and is directly correlated withthe cellular growth rate (Amann et al., 1995). Probes specific to rRNA (mainly 16S and 23S rRNA sequences)ได้กลายเป็นเครื่องมือมาตรฐานสำหรับสิ่งมีชีวิตรหัส (โอลเซ็น et al., 1986)คลิปปากตะเข้ oligonucleotide หลายได้ในเชิงพาณิชย์ว่าง และเลือกว่าจะใช้ หรือออกแบบใหม่ เฉพาะขึ้นอยู่ส่วนใหญ่ในการแอพลิเคชัน หาลำดับเป้าหมายอย่างต่อเนื่องนักวิจัยใช้เฉพาะจุลินทรีย์ที่เฉพาะเจาะจงในการเปรียบเทียบลำดับการใช้คอมพิวเตอร์ช่วยในโครงการฐานข้อมูล Ribosomal (RDP) (Maidak et al.,1996), ใน GenBank ในดีเอ็นเอ (Benson et al., 1999), หรือในแพคเกจซอฟต์แวร์ ARB (สถาบันของพลังค์ Maxเบรเมน เยอรมนี) สำหรับ specificity ดี เป้าหมายลำดับควรสั้น (15 ถึง 30 ฐาน) และมีอย่างน้อยสองถึงสามอื่นนิวคลีโอไทด์ ด้วย homologousลำดับของสิ่งมีชีวิตที่เกี่ยวข้องอย่างใกล้ชิด (DeLong1993) ทำงานช่วงบนใน situ hybridization อาศัยในคลิปปากตะเข้กัมมันตรังสีเพื่อเปิดเผย และตรวจสอบ probetargetไฮบริด (โอลเซ็น et al., 1986) ทำงานปัจจุบันrRNA ใน situ hybridization ใช้ฟลูออเรสเซนต์ชื่อนิวคลีโอไทด์ probes เกือบเฉพาะการ hybridization(ปลา) ความนิยมของเทคนิคปลาเนื่องจากมันเกิดประโยชน์กว่าการติดฉลากกัมมันตรังสีมีความไว ความเร็วของภาพแสดงเซลล์เดียว (โดยวิธี microscopy หรือ cytometricalอุปกรณ์), ความเสถียรของผลิตภัณฑ์ hybridizationความปลอดภัย ป้ายหลาย เวลาตรวจจับลดลง(หลายสี) และความสะดวกในการใช้ (ริชาร์ดสัน et al.,1991 DeLong, 1993 Swinger กทักเกอร์ 1996)สี fluorescein และ rhodamine มีบ่อยที่สุดused fluorochromes (DeLong et al., 1989;Amann et al., 1990; Manz et al., 1992; 1993; Wagneret al., 1994), but the CY3 dye, brighter and leading tolower non-specific fluorescence than the other fluorochromes,has recently been attracting more attention(Wessendorf and Brelje, 1992; Glockner et al., 1996;Zarda et al., 1997; Kalmbach et al., 1997a; Ouverneyand Fuhrman, 1999). In practice, FISH proceduresteps are: cell fixation, hybridization (specificity andstringency depend on hybridization temperature andtime, salt concentration, probe concentration andlength), post-hybridization washing (to removeunbound or non-specifically bound material) anddetection. Hybridized cells are usually detected byepifluorescence microscopy and a counterstain suchas DAPI (40,60-diamidino-2-phenylindole) or orangeacridine is used to determine the total number of cells.Depending on the concentration of targeted cells inthe sample and to increase resolution, FISH detectioncan be performed by means of flow or solid-phasecytometry. Flow cytometry enables quantification ofthe fluorescence intensities for each target-probehybrid (Fuchs et al., 1998).For TC, the development of a specific 16S rRNAprobe for this group is not possible, since the coliformgroup as defined by the water industry is a groupcontaining bacteria from genera that are phylogeneticallydifferent. As a result, probes (Table 3) weredeveloped for the Enterobacteriaceae family ratherthan for TC. The first, ENTERO (Mittelman et al.,1997) was developed for clinical detection of urinarytract infections. The second, ENT1 (Loge et al., 1999)was developed for application on wastewater samples.These two probes are different in their compositionand in the hybridization conditions. The ENTEROprobe is longer-25 bases with a C +G content of 48%compared to 17 bases and a 70% C +G for the ENT1probe. Research within the RDP (Maidak et al., 1996)showed higher specificity for the ENT1 probe forEnterobacteriaceae species than the ENTERO probe(Rompre´ and Baudart, NSERC Industrial Chair onDrinking Water, Ecole Polytechnique Montreal, personalcommunication).Sequences of rRNA target probes for the detectionof E. coli have also been published. The EC1531probe (Poulsen et al., 1994), complementary to a 23SrRNA sequence, is composed of 20 nucleotides with aC +G content of 55%. Shi et al. (1999) used thisprobe for the detection of E. coli after modifying thehybridization conditions, but no results were mentioned.The probe had only been used as a control inan experiment using a microcosm extract spiked withE. coli DNA. More recently, Regnault et al. (2000)developed the Colinsitu probe for the detection of E.coli and E. fergusonii in urine, water (rivers andsewage) and food samples. The Colinsitu probe complementsa 16S rRNA sequence and is composed of24 nucleotides (C +G content of 46%). This probeshowed a good specificity for the visualization of E.coli from the various media tested and has newly beenused with success for the detection of E. coli indrinking water samples (Delabre et al., 2001).The FISH technique appears to be a highly specificdetection method at a cellular level; however, it mayhave some limitations when applied to the detection ofnutrient starved bacterial cells disseminated in drink-ing waters. These limitations are linked to a lowbacterial ribosome content and thus to the smallnumber of 16S rRNA targets of these cells, whichinduces weak fluorescent hybridization signals(Amann et al., 1995; Lebaron et al., 1997). This isprobably why FISH is rarely described in the drinkingwater literature at this time. As a consequence, fluorescenceamplification systems, such as multipleprobing (instead of monolabeled probing in the conventionalFISH technique), interactive properties ofthe biotine–avidine complex and/or horseradish peroxydaseconjugated with fluorochrome–tyramidesubstratum,could represent a way to increase theintensity of the fluorescent signal given off by starvedhybridized cells (Lebaron et al., 1997; Scho¨nhuber etal., 1997). Recently, Prescott and Fricker (1999)reported the use of a peptide nucleic acid (PNA)probe for in situ hybridization and detection of E.coli in tap water. The hybridization results weresimilar to those yielded by the plate count method.A PNA is a synthetic nucleic acid in which the sugarbackbone is replaced with a peptide backbone. Theadvantages to using PNA instead of DNA probesinclude better resistance to nuclease attack, hybridizationindependent of salt concentration, more specificbinding and shorter probes sequences, to achievegreater sensitivity. Since the PNA probe binds morestrongly to the target site, hybridization reaction canbe completed in a shorter time (30 min). To ourknowledge, except for the E. coli PNA probe (Prescottand Fricker, 1999), none of these probes has beenused for coliforms or pathogenic bacteria monitoringin drinking water. Before they are accepted as asensitive coliform enumeration method, they mustfirst be subjected to a validation stage so that theprobe most suitable for the samples analyzed can bechosen and to optimize the hybridization conditions ifnecessary.Cell viability is a major question arising in theutilization of FISH for monitoring contamination of specificity of the oligonucleotidic probe and on thestringency of hybridization conditions used. Identificationof the target sequence remains tedious, however,and FISH cannot be applied to the detection of nonphylogeneticallyidentified micro-organisms, such ascoliforms. In this case, work can be done on Enterobacteriaceae,the nearest phylogenetically identifiedgroup that can be considered as a potential indicator offecal contamination.drinking water. It is established that the rRNA contentof the bacterial cells is correlated directly with thegrowth rate (Amann et al., 1995). However, the rRNAcontent of a bacteria may not completely reflect itsphysiological status. It appears that a small number ofrRNA molecules can remain for a relatively longperiod after the loss of culturability. S. aureus and E.coli rRNA were still detected 48 h after moderate heatactivation or UV irradiation (McKillip et al., 1998).Sheridan et al. (1998) still found E. coli 16S rRNA 16h after thermal inactivation at 100, 80 and 60 C.Finally, Prescott and Fricker (1999) exposed an E. colistrain to 1.5 mg/l of chlorine for up to 30 min. Usingthe PNA probe and the tyramide fluorescent amplificationsystem (TSA kit; Perkin Elmer Life Science,Ontario, Canada), they still detected bacteria immediatelyafter chlorination, while no cells were detected bythe plate counts or Colilert methods. Two weeks afterchlorination, 10% of the cells were still being detectedby the PNA probe-TSA kit. This can be viewed as anadvantage to the method in that non-culturable coliformscan be detected, or as a disadvantage in that deadcells might be enumerated. Further investigations onthe physiological state of the bacteria enumerated by

ผลลัพธ์ (ไทย) 3:

คุณใช้ ซึ่งวิธีการตรวจแบบ
กรดนิวคลีอิกลำดับ วิธีนี้ใช้
ความสามารถของกรดนิวคลีอิกเพื่อเนล อีกคนหนึ่งในวิธีที่เฉพาะเจาะจงมากซึ่ง
รูปแบบไฮบริด
) เป็นเฉพาะเพราะพวกเขาถูกสร้างขึ้นจาก และประกอบกับมี

เลือกลำดับกรดนิวคลีอิกซึ่งเป็นเอกลักษณ์ให้จุลินทรีย์ชนิดหรือ
กลุ่มฟิวส์ที่สามารถกำหนดเป้าหมายให้ DNA หรือ RNA
โมเลกุล ใช้ลำดับ ( 5S rRNA ( และ ยุค 23 )
ความสัมพันธ์ต่างๆ บนพื้นฐานของความแตกต่างของพวกเขาและพัฒนา (

) ตอนนี้เป็นตัวกำหนดก่อตั้ง ( แอเมิน et al . , 1995 ) .
อายุกว่า 2 , 500 ชนิดของแบคทีเรีย
เบส 16S rRNA ยีนเหล่านี้
เกี่ยวข้องในปัจจุบันข้อมูลมูลค่าสูงมากในระดับต่างๆ .
เปรียบเทียบลำดับทำให้มันเป็นไปได้ในการกำหนดพื้นที่อนุรักษ์ที่สมบูรณ์

ระดับการศึกษาที่แตกต่างกันและจากนั้นภายในเฉพาะ
ระดับเหล่านี้ เป้าหมาย จาก โดเมน สายพันธุ์ ของ
สนใจเป้าหมาย rRNA โมเลกุลระดับโมเลกุลสําเนา
ของแบคทีเรีย ซึ่งจะเชื่อมโยงกับ