DE102006048264B4 - Method for detecting specific nucleic acid sequences - Google Patents

Abstract

Method for carrying out detection of specific nucleic acid sequences using a test kit, having the following features:
The test set comprises a probe carrier plate (10) and a sample carrier plate (20),
The probe support plate (10) has at least one probe reaction surface (11),
According to a first alternative, a probe composition (15) which is liquid at the hybridization temperature is applied to at least one probe reaction surface (11),
According to a second alternative, the test set additionally comprises a container with a liquid probe composition and each probe reaction surface (11) is formed and coated so that the liquid probe composition is applicable,
The sample carrier plate (20) has at least one sample reaction surface (21),
Each sample reaction surface (21) is formed and coated so that a sample composition (25) can be applied whose sample nucleic acid sequences are immobilizable on the sample reaction surface (21),
The sample carrier plate (20) can be plugged onto the probe carrier plate (10),
- That at least one probe reaction surface (11) and a sample reaction surface ...

Description

The invention relates to a method for detecting specific nucleic acid sequences.

The DE 100 58 108 A1 shows a sample carrier with a lid. So that the sample does not dry, the lid is porous and moistenable. There is an indication that the sample carrier can also be used for hybridization. The reactions take place on the sample carrier. A defined arrangement of the reaction components is not required.

The DE 202 07 612 U1 relates to a device for detecting specific nucleic acid sequences. On a slide a variety of DNA samples are grid-shaped and immobilized. Detection of specific nucleic acid sequences occurs in a device in an automated manner. The device includes sealing means to form a hybridization space. Fluid media enters and exits the hybridization space and is constantly kept moving.

The WO 00/05336 A2 shows a device for the automated detection of specific nucleic acid sequences in liquid samples. Here, probe nucleic acid sequences are immobilized on a probe reaction surface. A hybridization space is formed around the probe reaction surface. It is an automated device. The temperature and humidity can be regulated.

The DE 693 23 720 T2 shows a device for detection of specific nucleic acid sequences. Between a slide and a cover, there is a fluid with a mix of the appropriate fluids to perform in situ PCR amplification. The lid mechanism is complexed to control the reaction conditions.

The US 2006/019 91 93 A1 relates to the automated sequencing of large quantities of DNA with microarrays using electrochemical methods. The probe nucleic acid sequences are immobilized in separate sections on the microarrays.

• • dass mindestens eine Gewebereaktionsfläche und eine Serumreaktionsfläche gegenüberliegend unter Bildung eines Reaktionsflächenpaares angeordnet sind, derart, dass zwischen der Gewebereaktionsfläche und der zugeordneten Serumreaktionsfläche ein vordefinierter Abstand vorliegt,
• • dass die Gewebeträgerplatte und die Serenträgerplatte Dichtmittel aufweisen, die in der Einsteckstellung um mindestens ein Reaktionsflächenpaar angeordnet sind.

From the EP 0 117 262 B1 is a test kit for the detection of antibodies in sera known. Furthermore, the Internet under the search term "Titerplane ^® " more references to the subject of the aforementioned patent. The known test set comprises a fabric carrier plate and a serenträgerplatte. The tissue carrier plate has a plurality of tissue reaction surfaces on which biological tissue is applied and immobilized. The serge plate has multiple serum reaction surfaces. The serum reaction surfaces are designed and coated so that sera can be applied in one step and labeled antiserum in a further step. The serge carrier plate can be plugged onto the fabric carrier plate in such a way that

• That at least one tissue reaction surface and a serum reaction surface are arranged opposite to form a reaction surface pair, such that a predefined distance exists between the tissue reaction surface and the associated serum reaction surface,
• • that the fabric support plate and the serge support plate have sealing means which are arranged in the insertion position around at least one reaction surface pair.

• – Die beiden Trägerplatten sind einzig und allein für die Immunologie vorgesehen.
• – Demzufolge dient die eine Trägerplatte dazu, biologisches Gewebe aufzunehmen.
• – Die andere Trägerplatte dient dem Auftragen der Seren.
• – Die Verfahrensschritte sind überhaupt nicht miteinander vergleichbar. Beispielsweise wird auf die Serenträgerplatte in einem Schritt das Serum aufgetragen. Dann steckt man die Serenträgerplatte mit der Gewebeträgerplatte zusammen. Nach einer Einwirkungszeit trennt man beide Platten voneinander. Die Serenträgerplatte wäscht man ab. Dann trägt man ein markiertes Antiserum auf die Serenträgerplatte auf. Es folgt ein nochmaliges Zusammenstecken der Gewebeträgerplatte auf die Serenträgerplatte, usw.

In other words, a test set is described which has two carrier plates. These carrier plates are adapted to be used as a sample carrier plate as set out in claim 1 or as a probe carrier plate as described in claim 1. The differences from the subject matter of claim 1 are:

• - The two carrier plates are intended solely for immunology.
• As a result, the one carrier plate serves to receive biological tissue.
• - The other carrier plate is used to apply the serums.
• - The process steps are not comparable at all. For example, the serum is applied to the serge plate in one step. Then you put the serenträgerplatte together with the tissue carrier plate. After an exposure time, separate both plates from each other. The serenträger plate washes off. Then apply a labeled antiserum to the serge plate. This is followed by a repeated mating of the fabric carrier plate on the serenträgerplatte, etc.

• • Auftragung der Probenzusammensetzung auf die Reaktionsflächen
• • Immobilisierung der Probennukleinsäuresequenzen auf den Reaktionsflächen
• • Auftragung der Sondenzusammensetzung auf dieselben Reaktionsflächen
• • Einbringung in einen Hybridisierungsofen
• • Herausnahme nach Ablauf der Hybridisierungszeit
• • Waschen der Trägerplatte
• • Auswertung der Probenträgerplatte.

From the DE 38 53 120 T2 For example, a test kit and method for detecting specific nucleic acid sequences is known. The test kit comprises a single carrier plate with a plurality of reaction surfaces. Furthermore, the test set includes a container with a probe composition. The procedure is as follows:

• • Application of the sample composition to the reaction surfaces
• Immobilization of the sample nucleic acid sequences on the reaction surfaces
• Application of the probe composition to the same reaction surfaces
• • introduction into a hybridization oven
• • Removal at the end of the hybridization period
• • Washing the carrier plate
• • Evaluation of the sample carrier plate.

• • Anstelle zweier Trägerplatten in Form einer Sondenträgerplatte und einer Probenträgerplatte wird nur eine Trägerplatte verwendet. Dies ist der wesentliche Unterschied.
• • Auf dieser einzigen Trägerplatte sind Reaktionsflächen angeordnet.
• • Auf einer Reaktionsfläche wird erst eine Probenzusammensetzung aufgetragen, deren Probennukleinsäuresequenzen man auf der Probenreaktionsfläche immobilisiert.
• • Auf der selben Reaktionsfläche trägt man die Sondenzusammensetzung auf.
• • Zur Stabilisierung der Reaktionsbedingungen wird hier eine zusätzliche gasdichte Hybridisierungskammer mit einer gesättigten Wasserdampfatmosphäre benötigt. Denn sonst würden die Probenzusammensetzung und die Sondenzusammensetzung eintrocknen.

In other words, the acquaintance agrees with the DE 38 53 120 T2 except for the following difference features with the objects of the following claims:

• • Instead of two carrier plates in the form of a probe carrier plate and a sample carrier plate, only one carrier plate is used. This is the essential difference.
• • Reaction surfaces are arranged on this single carrier plate.
• On a reaction surface, first a sample composition is applied whose sample nucleic acid sequences are immobilized on the sample reaction surface.
• • Apply the probe composition to the same reaction surface.
• • To stabilize the reaction conditions, an additional gas-tight hybridization chamber with a saturated water vapor atmosphere is needed here. Otherwise the sample composition and the probe composition would dry up.

The invention is based on the object, in the DE 38 53 120 T2 educate described method for the detection of specific nucleic acid sequences so that the handling is simplified.

This object is achieved by a method having the features of claim 1.

The advantages of the invention lie in the rapid and simple implementation of a detection of specific nucleic acid sequences. The test set used comprises a probe carrier plate and a sample carrier plate. The probe support plate has at least one probe reaction surface. According to a first alternative, a probe composition which is liquid at the hybridization temperature is applied to at least one probe reaction surface. This saves the user of the test set the application of the probe composition. Furthermore, the risk of erroneous application of the probe composition is excluded. The sample carrier plate has at least one sample reaction surface. Each sample reaction surface is formed and coated to accept a sample composition whose sample nucleic acid sequences are immobilizable on the sample reaction surface. The sample carrier plate can be plugged onto the probe carrier plate in such a way that at least one probe reaction surface and a sample reaction surface are arranged opposite one another to form a reaction surface pair. There is a predefined distance between the probe reaction surface and the associated sample reaction surface. When the probe carrier plate and the sample carrier plate are put together, the process of incubation begins simultaneously for all reaction surface pairs. The probe carrier plate and the sample carrier plate have sealing means, which are arranged in the insertion position to at least one reaction surface pair. Due to the sealant no hybridization chamber is needed with a saturated water vapor atmosphere. Because the sealants prevent the probe composition and the sample composition from drying out.

According to a second alternative, in contrast to the first alternative, the user himself still has to apply the liquid probe composition to the probe carrier plate. Therefore, with regard to the second alternative, on the one hand, a container with a liquid probe composition is an integral part of the test set and, on the other hand, the probe carrier plate without a probe composition already applied.

According to one embodiment of the invention, the probe compositions applied to the probe reaction surfaces are gel-like at room temperature. This embodiment relates to the subject of the first alternative, in which the probe composition is already applied to the probe support plate. This embodiment allows a factory-made production of prefabricated probe support plates, which are safe to handle at room temperature and lower temperatures. They can be held in any desired position in the room without the probe compositions of the individual probe reaction surfaces mixing with each other. Ready-to-use probe carrier plates with applied probe composition can thus also be packaged and shipped to customers. The industrial coating with probe compositions allows a maximum of quality assurance and prevents interchanging on the probe reaction surfaces.

According to another embodiment of the invention, which relates to the gel-like probe composition, the probe composition comprises agarose. Agarose is known as a gelling agent and is used in gel electrophoresis. The new use of the agarose serves as part of the probe composition to convert a liquid probe composition into a handling-safe gel. The advantage of the gelling agent agarose is that it is commercially available in different qualities, which are set to a certain temperature at which the gel becomes liquid. By selecting a quality with your condensing temperature just below the hybridization temperature, you can achieve a very good handling. As a result, agarose stands out over other gelling agents, such as starch, glycogen or polyacrylamide, particularly positive.

The invention will be explained in more detail with reference to an embodiment shown in the drawings. Showing:

1 a test set for the detection of specific nucleic acid sequences, with a probe support plate and a sample support plate, in perspective view;

2 the probe support plate of in 1 illustrated test sets, on average;

3 the sample carrier plate of in 1 illustrated test sets, on average;

4 the probe carrier plate and the sample carrier plate of the 1 illustrated test sets in the insertion position, in section.

The 1 shows a test set for the detection of specific nucleic acid sequences. The test kit includes a probe support plate 10 and a sample carrier plate 20 ,

The probe carrier plate 10 has several probe reaction surfaces 11 on. On every probe reaction surface 11 is a probe composition 15 which is liquid at the hybridization temperature.

The sample carrier plate 20 has several sample reaction surfaces 21 on. Each sample reaction area 21 is formed and coated so that a sample composition can be applied, its sample nucleic acid sequences on the sample reaction surface 21 are immobilized.

The sample carrier plate 20 is so on the probe support plate 10 pluggable that at least one probe reaction surface 11 and a sample reaction area 21 opposite to form a reaction surface pair 50 are arranged. In the insertion position lies between the probe reaction surface 11 and the associated sample reaction area 21 a predefined distance a before, as best 4 shows.

The 1 illustrates that the probe support plate 10 and the sample carrier plate 20 Have sealing means which in the insertion position by at least one reaction surface pair 50 are arranged. The sealants are based on the principle of the gap seal. Surfaces of the probe carrier plate 10 lie opposite surfaces of the sample support plate 20 and form a sealing gap. A sealing gap is formed by the drawn rectangle circumference 16 the probe carrier plate 10 and the drawn-in rectangle size 26 the probe carrier plate 20 , Opposing side surfaces 17 the probe carrier plate 10 dense with opposite side surfaces 27 the sample carrier plate 20 from. The same applies to the side surfaces 18 the probe carrier plate 10 and the side surfaces 28 the sample carrier plate 20 ,

The probe carrier plate 10 and the sample carrier plate 20 have a conventional way of securing against interchanging the mating position. Serve for this purpose on the probe support plate 10 attached formations 19 with a geometric shape and corresponding recesses 29 in the sample carrier plate 20 ,

The on the probe reaction surfaces 11 applied probe composition 15 is gelatinous at room temperature. This facilitates the transport from the manufacturer of the test set to the customer, as already explained under the point of the benefit label. Furthermore, the gel-like probe composition facilitates handling.

• • Auftragung der Probenzusammensetzung 25 auf die Probenreaktionsflächen 21.
• • Immobilisierung der Probennukleinsäuresequenzen auf den Probenreaktionsflächen 21.
• • Zusammenstecken der Sondenträgerplatte 10 mit darauf aufgebrachter Sondenzusammensetzung 15 und der Probenträgerplatte 20. – Dieser Verfahrensschritt bezieht sich zum einen auf das dargestellte Ausführungsbeispiel. Die Sondenträgerplatte 10 mit darauf aufgebrachter Sondenzusammensetzung 15 ist die Sondenträgerplatte 10 mit der bereits vom Hersteller des Testsets aufgebrachten gelartigen Sondenzusammensetzung 15. – Ferner bezieht sich dieser Verfahrensschritt auf das nicht dargestellte Ausführungsbeispiel, bei dem zu Anfang eine Sondenträgerplatte 10 ohne bereits aufgebrachte, gelartige Sondenzusammensetzung vorliegt und bei dem der Hersteller ein Behältnis mit einer flüssigen Sondenzusammensetzung mit dem Testset integriert hat. Der Anwender hat daher noch den Schritt des Auftragens der flüssigen Sondenzusammensetzung selbst auszuführen.
• • Einbringung in einen Hybridisierungsofen.
• • Herausnahme nach Ablauf der Hybridisierungszeit.
• • Lösen der Sondenträgerplatte 10 von der Probenträgerplatte 20.
• • Waschen der Probenträgerplatte 20.
• • Auswertung der Probenträgerplatte 20.

The method for carrying out detection of specific nucleic acid sequences using the test kit shown in the drawings comprises the following steps:

• • Application of the sample composition 25 on the sample reaction surfaces 21 ,
• Immobilization of the sample nucleic acid sequences on the sample reaction surfaces 21 ,
• • Connecting the probe support plate 10 with probe composition applied thereto 15 and the sample carrier plate 20 , - This process step relates firstly to the illustrated embodiment. The probe carrier plate 10 with probe composition applied thereto 15 is the probe support plate 10 with the gel-like probe composition already applied by the manufacturer of the test kit 15 , - Further, this method step refers to the embodiment, not shown, in which initially a probe support plate 10 without an already applied gel-like probe composition and in which the manufacturer has integrated a container with a liquid probe composition with the test kit. The user therefore still has to carry out the step of applying the liquid probe composition itself.
• • introduction into a hybridization oven.
• • Removal at the end of the hybridization period.
• • Loosen the probe carrier plate 10 from the sample carrier plate 20 ,
• • Washing the sample carrier plate 20 ,
• • Evaluation of the sample carrier plate 20 ,

Details of the aforementioned method are described below using the example of the detection of Escherichia coli. Escherichia coli is found in human urine and is the most common pathogen of the human urinary tract.

A probe carrier plate is used 10 and a sample carrier plate 20 , The square probe reaction surfaces 11 and sample reaction areas 21 each have an edge length of 5 mm. This results in a surface size of 25 mm ² . The distance a, the best in 4 is illustrated, is 1 mm. 1 illustrates that the probe reaction surface 11 via a hydrophobic interface 12 and the sample reaction area 21 via a hydrophobic interface 22 are separated from each other.

In the following, the industrial application of a probe composition to the probe support plate will first be described, wherein the applied probe composition is gelatinized for better handling at room temperature.

On probe reaction surfaces 11 a probe carrier plate 10 each 25 ul of a probe composition 15 Pipette. The probe composition has 50 ° C, the probe support plate at room temperature.

• – 50 pg/ml Nukleinsäuresonde ESCO47, am 5'-Ende FITC markiert Sequenz: GCT GGT AAC GTC AAT GAG C Bezugsquelle: Thermo Hybaid, Ulm
• – 0,8% Agarose (Art. Nr A9539, SIGMA-Aldrich)
• – 30% Formamid
• – 900 mmol/l NaCl
• – 20 mmol/l Tris-HCl
• – 0,01% SDS (Sigma-Aldrich)
• – ad 100% Reinstwasser

The probe solution is composed as follows:

• - 50 pg / ml nucleic acid probe ESCO47, at the 5'-end FITC labeled Sequence: GCT GGT AAC GTC AAT GAG C Source of supply: Thermo Hybaid, Ulm
• - 0.8% agarose (Art. No A9539, SIGMA-Aldrich)
• - 30% formamide
• - 900 mmol / l NaCl
• 20 mmol / l Tris-HCl
• - 0.01% SDS (Sigma-Aldrich)
• - ad 100% ultrapure water

Immediately after touching the probe composition 15 with the probe reaction surface 11 The wetting drop solidifies to gel. The finished probe carrier plate 10 can be shipped at room temperature. Until use, a cool and dark storage is preferable.

For example, a user who has purchased the previously mounted probe support plate will now perform the following steps.

First, the sample composition 25 to create. Escherichia coli is considered to be significant for the presence of a specific urinary tract infection from a bacterial count of 10,000 per μl in the urine. Based on this germ count in a urine sample, the urine sample is enriched by centrifugation 1:10 to a bacterial count of 100,000 per μl to increase the detection limit, carries 10 μl of this sediment on a sample reaction surface 21 and let it dry. There are then 1 million copies of the bacterium. The enriched urine corresponds to the sediment of the centrifugation.

By dipping the sample carrier plate 20 in methanol and subsequent drying, the bacteria on the sample reaction surfaces 21 fixed. The prepared sample carrier plate 20 can be transported at room temperature or stored for a long time in a cool and dark place.

The user who prepared the sample carrier plate prepared by him 20 and the industrially coated probe support plate 10 has cooled to room temperature. 15 minutes temperature adjustment time suffice. Uncooled sample carrier plates or probe carrier plates can be processed immediately. By plugging together the sample carrier plate 20 and probe carrier plate 10 arise reaction surface pairs 50 , The space between the reaction surface pairs becomes exactly the same as the probe composition 15 filled. Due to the area size of 25 mm ² and the distance a of 1 mm, a volume of 25 μl is available. This volume corresponds to the amount of probe composition to be pipetted.

The assembled probe carrier plate 10 and sample carrier plate 20 is placed in the hybridization oven at 48 ° C for 60 minutes. Using an agarose gel melting at 40 ° C, the now liquid probe composition fills 15 the space between the reaction surface pairs 50 completely off. Capillary forces now fix the probe composition 15 in the space between the respective reaction surface pairs 50 , By diffusion and convection, the contact and specific binding between the probe nucleic acid sequence and the complementary nucleic acid sequence of the single-stranded RNA of the bacterium can take place in the liquid medium.

To 60 Minutes become the assembled probe carrier plate 10 and sample carrier plate 20 removed and separated while warm. The sample carrier plate 20 rinsing with pre-tempered wash solution (50 ° C) in the torrent.

• – 112 mmol/l NaCl
• – 20 mmol/l Tris-HCl
• – 5 mmol/l Na-EDTA
• – 0,01% SDS

For the washing solution, the following composition is used:

• - 112 mmol / l NaCl
• 20 mmol / l Tris-HCl
• 5 mmol / l Na-EDTA
• - 0.01% SDS

Subsequently, the sample carrier plate 20 Washed three times for 5 minutes in 50 ° C warm wash buffer and rinsed with distilled water. The washing steps are required to substantially remove unbound probe nucleic acids.

The wet sample carrier plate 20 can now be covered with glycerin and coverslip. When covered, the sample carrier plate 20 be dried mechanically. Until the evaluation, it should be stored in a cool and dark place, if not immediately.

The evaluation is carried out under a fluorescence microscope (eg Zeiss Axiostar with Fraen transmitted-light fluorescence illumination A.F.T.E.R 480 nm, magnification 400 to 1200-fold, reference numeral: Apertur> 0.9).

In the presence of Escherichia coli, these are clearly visible as green glowing rod bacteria against a black background. Other bacteria that do not have the complementary nucleic acid sequence to the sample can not bind the dye and will not shine.

• • Die flüssige Sondenzusammensetzung des Behältnisses ist wie zuvor beschrieben zusammengesetzt, jedoch ohne das Gelierungsmittel Agarose.
• • Um die Sondenzusammensetzung aufzutragen, legt man die Sondenträgerplatte 10 mit den Sondenreaktionsflächen 11 nach oben auf eine Arbeitsplatte. Beim Zupipettieren der kalten, flüssigen Sondenzusammensetzung sind Erschütterungen unbedingt zu vermeiden, da sonst die Sondenzusammensetzungen der einzelnen Sondenreaktionsflächen 11 ineinander verlaufen können. Die Auftragepositionen der einzelnen Sondenzusammensetzungen sind zwingend zu dokumentieren und die Zuordnung zu überprüfen.
• • Da die Sondenzusammensetzung flüssig ist, muss die Handhabung beim Zusammenstecken der Probenträgerplatte und der Sondenträgerplatte und das Einbringen in den Hybridisierungsofen sehr sorgfältig erfolgen.

Deviating from the illustrated embodiment, it is possible that the test set for the detection of specific nucleic acid sequences a probe support plate 10 without already applied gel-like probe composition, a sample support plate 20 and a container having a liquid probe composition. As differences from the previous embodiment are called:

• The liquid probe composition of the container is composed as previously described, but without the gelling agent agarose.
• • To apply the probe composition, place the probe support plate 10 with the probe reaction surfaces 11 upwards on a worktop. When pipetting up the cold, liquid probe composition, shocks must be avoided, otherwise the probe compositions of the individual probe reaction surfaces 11 can run into each other. The application positions of the individual probe compositions must be documented and the assignment checked.
• • Since the probe composition is liquid, the handling of the sample support plate and the probe support plate and the introduction into the hybridization furnace must be handled very carefully.

LIST OF REFERENCE NUMBERS

10

Probe carrier plate

11

Probe reaction surface

12

Separation surface (hydrophobic)

15

probe composition

16, 17, 18

sealing surfaces

19

Anformung with a geometric shape

20

Sample support plate

21

Sample reaction surface

22

Separation surface (hydrophobic)

25

sample composition

26, 27, 28

sealing surface

29

Recess with geometric shape

50

Reaction surface pair

a

predefined distance

SEQUENCE LISTING

Claims (3)

Method for carrying out detection of specific nucleic acid sequences using a test set, having the following features: the test set comprises a probe support plate ( 10 ) and a sample carrier plate ( 20 ), • the probe support plate ( 10 ) has at least one probe reaction surface ( 11 ), according to a first alternative, on at least one probe reaction surface ( 11 ) a probe composition ( 15 ), which is liquid at the hybridization temperature, according to a second alternative, the test set additionally comprises a container with a liquid probe composition and each probe reaction surface ( 11 ) is formed and coated so that the liquid probe composition can be applied, 20 ) has at least one sample reaction surface ( 21 ), • each sample reaction surface ( 21 ) is formed and coated so that a sample composition ( 25 ) whose sample nucleic acid sequences on the sample reaction surface ( 21 ) are immobilisable, • the sample carrier plate ( 20 ) is on the probe support plate ( 10 ) pluggable, That at least one probe reaction surface ( 11 ) and a sample reaction surface ( 21 ) opposite to form a reaction surface pair ( 50 ) are arranged such that between the probe reaction surface ( 11 ) and the assigned sample reaction surface ( 21 ) a predefined distance (a) is present, - that the probe support plate ( 10 ) and the sample carrier plate ( 20 ) Sealant ( 16 . 17 . 18 . 26 . 27 . 28 ), which in the insertion position around at least one reaction surface pair ( 50 ), the process steps are: application of the sample composition ( 25 ) on the sample reaction surfaces ( 21 ), - immobilization of the sample nucleic acid sequences on the sample reaction surfaces ( 21 ), - putting together the probe carrier plate ( 10 ) with a probe composition applied thereto ( 15 ) and the sample carrier plate ( 20 ), - introduction into a hybridization oven, - removal after the end of the hybridization time, - release of the probe support plate ( 10 ) from the sample carrier plate ( 20 ), - washing the sample carrier plate ( 20 ), - Evaluation of the sample carrier plate ( 20 ). Method according to claim 1 according to the first alternative, in which the on the probe reaction surfaces ( 11 ) applied probe composition ( 15 ) is gelatinous at room temperature. The method of claim 2, wherein the probe composition ( 15 ) Has agarose.

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