JPH01500008A - A method for tracing the ADN sequence of retrovirus HTLV3-LAV in seropositive individuals and a kit for its implementation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Follow-up of the ADN sequence of retrovirus HTLVII [-LAV in seropositive individuals. 7) Field of invention for trace method and its implementation The present invention generally provides lymphocyte T4 ADN cells by special use of conventional molecular hybridization techniques. A method for coloring the ADN sequence of retrovirus HTLVI [[-LAV] It is related to. Such methods are routine for corresponding sequences in seropositive individuals. Applicable to detection work. The method according to the invention provides a method for detecting lymphocyte T4 genomic information. and the direct release of a virus in its free state or in its conformational form in humans. make it possible to prove the existence (or absence) of a person. The present invention also provides for carrying out the method. The purpose is also to create a box for

In recent publications, the retrovirus HTLVII [-LAV was studied and It has been shown that this is the cause of IZ disease. Related references in this field include Sha Ugh.

M, (SHAW, G, M,,), Burn Bee, Ecchi.

(HAHN B, H,,), Alya Nis, Kay, (ARYA S, Ni,,), GRooPMAN J, E, (GRooPMAN J, E,,), Galo C, (GALLOR, C,) and WONG-3TAAL, F. , F., ) (1984), “Human T-cell leukemia in acquired immunodeficiency disorders. "Molecular characterization of I [[(HTLV-III)"] Science e), '126-1165-1171.

Retrovirus HTLVI [[-LAV is an RNA virus, but lymphocyte Upon integration into T4, this allows simple AD to function due to its reverse transcriptase enzyme. N state and double chain state! (However, at this level it is still in the host genome.) It is not very clear whether it exists in free form or in an integrated state. It has already been demonstrated that the transition to , (HIR3CHM,S,) and Cabranjou. Sea, (KAPLAN, L , C,) Co-author (1985) Rhi) Treatment for type T mumpsysis ■ Outlook”Annals of Internal Medicine Internal Medicine, 103.750-755)).

The presence of the virus at this moment is much more important than between RNA molecules. Detection using the ADN-ADN molecular hybridization technique, which is easy, has already been done. It has been proposed. However, this technique cannot confirm the presence of viral sequences. In practice, this cannot be done with sufficient certainty. A positive result is very special. It is already true that this could only be determined in a very small number of other cases. It is certified. In other words, when detecting at the peripheral blood mononuclear cell level, AIDS infected person 1 Only one of the six people could be identified. [Shaw G, M, ( SHAW G, M, ), Burn Bee, H.

(HAHN B, H,,), GROOPMAN J, I+, (GROOPMAN J, E, ), Progy Varnish.

(BRODERS,,), Wong Goose Tail F.

(WONG-3TAAL F,,), and Garo Earl.

C-, (GALLOR, C,), (1985) HTLVI in French organizations [Molecular characterization of type I ADN sequences] [Atlanta AIDS International Symposium, details] The idea underlying the invention can certainly be used in a reliable manner for practical needs. Common molecular hybridization techniques were applied to seropositive individuals, with adaptations made to It is to be used as such. In fact, if a human comes into contact with this virus, AIDS This person becomes seropositive even if he or she does not develop the disease. Even more recent research shows that This is important because we know that a slow and gradual progression to AIDS disease can occur. The situation is fraught with danger. Therefore, due to the need for prevention, HTLVI[[-LAV highly desirable. Although it has already become a classic, one person is seropositive. There are tests to prove this or not. However, to the best of the applicant's knowledge, Additional information that can support the presence of a viral condition in such seropositive individuals There is no method, and you can certainly consider using normal culture methods, but these Testing for HTLVI [I-LAV] is too time consuming and expensive. In the case of viruses, these are ejective.

Summary and summary of the invention The purpose of the present invention is to determine the progress of seropositive individuals towards the disease. Follow-up methods in seropositive individuals with clear results that are of vital importance in The aim is to answer the above-mentioned practical needs by presenting the law as an allegory.

Therefore, the present invention is characterized by applying a general molecular hybridization technique including the following steps. The ADN sequence of retrovirus HTLVIII-LAV in seropositive individuals It concerns tracking methods.

a) Collect blood from the subject, separate white blood cells from it by a known method, and collect lymphocytes A. In the step of obtaining DN, this lymphocyte ADH is added in a unit amount of at least 30 μg. used.

b) transfer such amount of lymphocyte ADN to a non-nicked control within the viral ADN sequence; The stage of treatment with limiting enzymes.

C) The medium of step b) containing the virus in free or integrated form has a surface area of 1 Passed through a nitrocellulose filter that can capture up to 80 μg of ADN per CD. stage.

d) Convert the product remaining in the filter to the cloned HTLVn[-LAV virus. contact with one sonde, which is a sonde marked with a The sonde uses the ADN of the virus, which does not have the encryption part for the virus envelope region. It is a subcloned fragment, and the marking is ADH1 of the sonde. Activity as close as possible to the 5°10'cpmO value per μ, especially ADNl pg It is possible to give an activity of 5-108 to 22-1O9ep per unit.

e) reading the presence of the characteristic horizontal stripes of the virus by known methods.

Molecular hybridization techniques are well known to those skilled in the art and are therefore detailed here. In the case of retrovirus HTLVI [1-LAV, the above-mentioned Shaku (S HAW) You can refer to the paper by others (1985). To the needs of the present invention An adaptation of this technique to This can be understood from examples.

Description of examples In the first step of the method, the subject's peripheral blood is collected, which is then purified using known methods. Separate blood cells. ) (after hydrolysis using a suitable enzyme such as ba i) According to the characteristics of the present invention, the filter used in step C) Utilize as much lymphocyte ADN as possible that is compatible with the patient load. This unit amount is 30 μg or more of ADN. In practice, amounts in the range 30-50 μg should be used. Can be done. A sufficiently reliable response cannot be obtained with values below 30 μg. In addition If it exceeds 80 μg, too much blood must be collected. On top of that , C) The usage limit of the stage filter is reached. Lymphocytes between 30μg and 50μg A unit amount of ADN is readily obtained from about 10 million cells of a subject's blood.

As is customary in this technique, these unit quantities are e.g. are deposited into each gel well.

In step b) of the method according to the invention, said amount of lymphocyte ADH is A restriction enzyme selected so that it does not have a recognition site within the ADN sequence of a common virus will be processed. The effect of this is that free virus or its various conformations Corresponds to the seat. A small number of horizontal stripes were captured by large-sized autoradiography. The goal is to concentrate the final signal. To the needs of the method according to the invention As an example of a matching enzyme, first, it is known that it does not cut inside the viral sequence. I must name the XbAI enzyme that is present; Burn, H.

(HAHN B, H,,), SHAK G, M, (SHAW G, M,,) , ARYAS, K. POVICM,) GALLOR, C, (GALLOR, C,) and Wong -Stal F, (WONG-3TAAL F.

), co-author (1984), “Molecular cloning of the HTLVI virus associated with AIDS.” See J.N.A.T., 312; 166-169. However, other comparable enzymes can also be used.

In step C) of the method according to the invention, as high a density as possible per d of surface is applied. A highly sensitive nitrocellulose filter is used. of this filter The properties are compatible with the unit amount of lymphocyte ADN used in step a). . The filter should have at least 30 pg, preferably 80 pg per d of surface area. It is possible to capture up to ADN. In practice, Millipore ) filter, or even better, Sartorius Stirisocha and Stur (Sartorius 5chleicher & 5chull) BA85 A filter can be used. In this latter case, the AD of the H9V3 culture was tested. Preliminary experiments using H It follows that up to g can be detected using the technique described here. It is shown. In other words, there are approximately 104 molecules of ADN, or currently 500 molecules. This means that only one infected cell can be detected.

In step d), the envelope region, which is inherently variable in various separations, is A subcloned fragment of the virus's ADN without the cryptographic part. One sonde is used. In practice, the central part of the genome and the 5' end Using a sonde consisting of a fragment 5stl-5' of λBH-10 that does not correspond to Good results were obtained. Such sondes are known to experts in the field. It is something. In this regard, European Union published as No. 0173529 Another example of the method according to the invention can be found with reference to the patent application specification. According to a very important feature, the marking is 5.l/μg of sonde DNA. As close as possible to the value of Q’cpm, preferably 5.10” to 2 per μg of DNA. ・It is not possible to provide an extremely high specific activity of lQ'cpm. However, in practice, such activity can be achieved using a large number of in-shakes of small size. dC using marking system and Klenow polymerase Obtained by TP Markin; or 800C! J 1 mole of two radioactive elements Marking by movement of a nick that involves the intervention of element (dCTP + dATP) [nick It can also be obtained by ``translation''. Amersham sham) low temperature kit N5500 and markings PB10384 and PB10 385 - see examples below).

The best results were achieved by marking at 32p via a single radioactive element (dCTP). This allows for methods using synthetic oligonucleotides as an in-shake method. obtained from marking the sonde. For routine work, use commercially available Amel. Approximately 1-2X10 using a kit equivalent to sham (Amersham) ' A specific activity of cpm/μg was obtained. Similarly, under these conditions, this The method uses a very small amount of sonde (approximately 100,100n for each marking). However, extremely high purity is required compared to protein (nitrogen-containing organic matter) contaminants. It has also been confirmed that this is not the case. Therefore, the method is easier to use and cheaper. It is.

Color development takes place in a known manner by autoradiographic exposure. Implementation of the invention In some cases, an exposure period of a few days, for example 4 days, is sufficient for reading and therefore tracking. It has been confirmed that there is. Best results with ultra-sensitive autoradiography The film was obtained using Amersham.

The method according to the invention using the technique of molecular hybridization includes, inter alia, the following main steps: You can

(1) Phenol after proteolysis by proteolytic enzyme (proteinase) K Extraction of long-chain lymphocyte ADN with a Le-chloroform mixture.

(2) Weighing of the obtained ADN by spectrometry and its measurement on an agarose minigel. Confirmation of length and degradability by nuclease test.

(3) Exceeding 5 units per μg of ADH to be degraded by restriction enzyme XbaI hydrolysis.

(4) Restriction flag on 0.8% agarose gel in transfer buffer 10X SSC Extension of ment.

(5) 32PdC at 800 cu per mmole for 250/jg Ci Sonde by nick movement with TP+dATP or 32PdCTP only markings.

(6) 10 mg ADN of salmon semen denatured by sonication at 67°C for 48 hours /mf added pH = 7.5.50mM Tris-Hcj! ;50mMEDT A, 0°1% SDS, IOX Denhardt, 5xssc buffer Hybridization of filter and modified sonde in liquid.

(7) Final wash with 1x SSC and 0.1% SPS buffer.

(8) Dry the filter and install two reinforced screens with the sensitive film Its inclusion in a cassette with.

Note that autoradiography is performed at -70°C.

(9) Color development of horizontal stripes photographed by autoradiography.

αω　As a measure of the size of ADN) (indI[[ Autoradiography was taken on the proof plant form using diλ. Reading horizontal stripes.

Figure 1 of the attached drawings shows (1) seropositive cases > 0 (2) seropositive cases > 0 The radiographed horizontal stripes obtained from case and H9V3 cultures are shown. : In the last case () indicates a multiple integration site.

Thus, in seropositive individuals, the following baI fragments were observed: Ru; - A large amount corresponding to high molecular weight ADN that does not penetrate into the gel at all or hardly penetrates into the gel. Large fragment.

- A fragment of approximately 13 kb corresponding to a circular, monotonically cleaved virus.

-A fragment of approximately 9 kb corresponding to the m-type virus.

In comparison, under the same conditions, the ADN of H9V3 corresponds to a large fragment. A signal with a blurred weight limit showing how the virus multiclonally integrates. Gives one large size signal.

The photograph in Figure 1 showing the three migration channels of the gel was observed on autoradiography. These signals embody the results of the method according to the invention. are doing. In the case of H9V3 series, gel wells are placed in the region of ADN with large molecular weight. There is a weak signal that is difficult to penetrate; the main part of the signal is a diffuse patch in weight. Represented by dots (these dots are densely packed; they represent cells in culture). This is because each virus contains about 50 to 100 virus particles. ): Said exhibition In general, viral integration at the human ADN level occurs in a polyclonal manner (insertion site). This is due to the fact that there are many For channel 1, this means that It concerns seropositive individuals who are positive for the signal. autoradiography There are three horizontal stripes photographed; one of them does not penetrate into the gel well. Located within ADN with large molecular weight; autoradio with sizes of approximately 13kb and 9kb The two horizontal stripes imaged are probably two different conformations (superconvoluted and superconvoluted). It corresponds to the free form of the virus under the relaxed and/or relaxed form. some are extremely positive (This is the case shown in Figure 1), and their horizontal stripes are particularly clear. Notable and immediately visible after 4 days of autography. For channel 2, this to those who are seropositive but negative for the signal (even after long exposure). It is related to

These results demonstrate that the reading of the results obtained by the method according to the invention is to reveal the presence of HTLVII[-LAV virus in sexually transmitted individuals. Moreover, it shows that it can be done in a reproducible manner.

Nine of the total 14 people tested showed viral sequences. These 9 Four of the positive cases showed stronger horizontal stripes and were therefore recognized as strong positive; 5 showed only weak horizontal stripes, even after long exposures: these strong The difference in size is independent of the amount of ADN deposited in each well and probably exists. It seems to reflect the number of copies of the virus.

In an advantageous variant of the method according to the invention, undisassembled) (b) a.Both specimens of those degraded by enzymes such as I are deposited simultaneously on the gel. let The typical results thus obtained are shown in FIGS. 2 and 3.

It is similar to Figure 1, but it includes both disassembled and undisassembled specimens. Looking at Figure 2, we can see that the obtained signals are a large fragment and an integrated 13 kb signal. It can be seen that they correspond to a combined fragment and a free fragment of 9 kb.

In such embodiments, the first step is to determine the presence of virus within the undigested specimen. This was confirmed after disassembly.

Figure 3 shows the characteristic horizontal stripes of l3kb and 9kb on the 10th and 1st month, respectively. shows the effect of exposure duration.

Using an embodiment with double specimens (one that is unresolved and one that is said to be unresolved) The method based on the present invention was applied to 25 seropositive subjects. Each The test was repeated twice. The results are summarized in the table below. In this table -rT4/Te blood sampling" is the ratio of lymphocyte T4 to lymphocyte T day, and the measurement The determination is made at the time of blood draw and constitutes an assumption that the subject is seropositive. It means to be.

-rHBS/HBC antigen j indicates the presence or absence of an antigen corresponding to viral hepatitis. means.

The other legends in this table are general. The results show that 16 people or 64% of the total number tested are positive. You can confirm that.

A survey conducted on 25 people with M blood The fact that the observed horizontal stripes match well with the viral sequence may be due to the following reasons. More shown.

- The obtained signal is in the above-mentioned state after hybridization with a specific sonde.

Horizontal stripes of the expected size are observed.

- These horizontal stripes are similar to those of the control culture.

In the case of a control ADH taken randomly from the population and degraded by Xba I, No horizontal stripes are seen. Horizontal stripes are also not seen in cases of true AIDS under the same conditions: As already pointed out, detection of the virus by this method is not practical in patients. No (in this case the amount of T4 cells is very low and the main part of the virus is in the RNA0 form) ).

The observed horizontal stripes are not a result of contamination of the sonde with phage λ, but are due to Xbal. When degraded, phage λ has been demonstrated in this type of control degradation. 23.9 kb and 24.5 kb, which are much larger than those observed. gives two restriction fragments. In contrast, lymphocyte ADN of seropositive individuals >0 The presence of free viral sequences within the AD of these individuals not resolved on the gel Proved by moving H and crossing the sonde with the corresponding channel. ; in most cases there is a strong signal within the undegraded ADN (integrated sequence) One signal was obtained, and another much weaker signal was obtained within the horizontal stripes of the molecular weight of ylokb ( free form) is obtained.

The invention also includes kits, boxes or bags containing the necessary components for carrying out the method. That is the purpose. The unit quantities of these components vary depending on the number of specimens planned to be processed. . The kit according to the present invention mainly contains the following: - LAV sonde, e.g. corresponds to subcolon λBH-5 from which phage λ has been excised sonde.

ADN preparation of phage λ digested by Xba I used as a control.

One-enzyme digestion buffer The formulations that can be used for marking are Amersian France (A Mersham (France), Avenue des Ais, Canada 91944, response Yuri France (Anemu du anada 91944Les Ul It is commercially available from France). - Translation and enzyme buffers added to the cryogenic kit N5500) - αP dCTP (PB10385) and αP32dATP (PB1038 4), 800 ci/m mole (250 μCi each).

Low-temperature derivatives of marking (e.g., currently available at Organics) 12A Avenue du President Salvador Arundu (Aven uedu President 5alvador A11ende)9440 2 Vitry-sur-3ain France (Vitry-sur-3ain The basic sulfonation product commercialized by King) can also be used in the same way. As a derivative type, low temperature marking or H Normal site hybridization using radioactive markings as in 3 can also be performed.

The invention is more clearly illustrated by the complete Example 8.

example: A total of 'lQmIt' of blood is collected in 5% EDTA in saline. blood After centrifugation at 1500 rpm for 5 minutes, the plasma was removed by suction with a vacuum pump. Ru. Cell lysis of red blood cells was performed using 50ml of SLR buffer. (Tr i s 10m Adding M, Mg (!!25mM, NaC1!10mM) to fresh precipitate Homogenization is obtained until hemolyzed state and then 5-1 at 2000 rpm. A 0 minute centrifugation is carried out: the float is then sucked out with a vacuum pump.

A solution of proteolytic enzyme (brotinase) K was prepared at a final concentration of 0 and 1 mg/ml. Prepared in E buffer (Tr i 5-EDTA). Precipitate obtained previously was redissolved in 3 m SLR buffer and further dissolved in 5 LBli buffer (Trt s 10mM, EDTAlomM, NaCf50mM 5DS0.2%) and 1 redissolved in 3 mg of proteolytic enzyme per tube; A block was obtained, which was homogenized and then slowly stirred at 42°C overnight. Stirred (45-50 rpm in orbital water solution) * The phenol used for extraction is saturated with TrisO, 2M, pH=7.6.

The first extraction consists of phenol 3/4. Isoamide of chloroform 1/4 and 1/24 The upper aqueous phase is removed with a pipette and then the second The extraction is prepared with phenol chloroform; then the two phases are separated and the ADN is The lower organic phase is removed with a syringe. Finally, the third extraction It is done to remove phenol + isoamyl acid with loloform.

In the remaining group, ADN added 300all of Na0443M and heated in anhydrous evaporator at -20°C. It is precipitated by supplementing with 59 ml of alcohol. Ethanol with a pipette The removed and precipitated ADN is recovered by inverting the pipette: absolute ethanol. Wash it many times.

ADN is resuspended in 2-4 ml TE at 4°C for several days. This is DO2 60, and the size check was performed on a 0.7% agarose minigel. , coloring is done with ethidium bromide in TEA buffer.

Restriction that the amount of ADH indicated for each specimen exceeds 5 units per μg of ADN Decomposed by the enzyme Xbal. Restriction fragments were then added to 0.8% horizontal agarose. spread on a base gel.

After transfer, the gel was depuriner (H(10,25M) , denatured (NaC/!IM in NaCAIM) and neutralized with N at pH-7.0. a Tri, so, 5> at Cf1.5M, transfer buffer is 10x SSC, nitrate The transfer of cellulose onto the filter takes place at night. Finally dry the filter The ADH was then fixed in an oven at 80° C. for 6 hours.

The sonde used consists of the fragment 5stI-5' of λ-BH-10. . This is a nick- Marked by translation.

The filter is placed in an orbital water solution in a sealed plastic return path containing: Precrossed at °C = 25 ml buffer 5xssc (1 hour) then 5xSS C, 10x Denhardt (D), 0.1% SDS (1 o'clock ), then denatured in 5X SSC, 10XD, 1mM EDTA and 0.1% SDS. ADN 100μg/m1 of sonicated salmon semen was added (overnight). ).

Crosses with modified sondes were incubated in lQmA (5 XSSC1IOXD, 0.1% SDS 5mM EDTA, 50mM Tri Performed in s-H (1! pH=7.5 and ADN 10 mg/mA).

After crossbreeding, the filters are successively washed in the following ports: (in a 66°C case) 250mA); 5xssc, 10XD, 0.1%SDS, 0.1%NaPP1 for 30 min: then 3x SSC, 10xD, 1% SDS, 0.1% NaPPI 0 min; then 15 min with 2xSSC%IXD, 0.1% SDS, 0.1% NaPP1 and buffer l5SC, IXD, 0. 1% SDS, 0. 1% Na PP i (follow up with a counter) for the required time.

The filter is then dried under a lamp and heated with water. )3? fixed on T1 paper and wrapped in plastic film, then two reinforced strips Autoradiograph at -70°C for 1 to 30 days in a clean cassette. It is set aside for A.

Autoradiography was developed for 5 minutes, washed with water, allowed to settle for 5 minutes, It is washed again and placed in the dryer. Horizontal stripes photographed by autoradiography The size is the Hindl [[size expressed in fragments] of the ADN of phage λ. Calculated based on the scale.

The examples detailed above are illustrative of the method according to the invention and are not meant in a limiting sense. It's not something you have.

Variants of the working modality can also be developed using disassembly or non-disassembly embodiments (double specimens). As mentioned, it can be provided by experts in the field.

Thus, the present invention allows for the detection of trace amounts in seropositive individuals in vivo and under actual realistic conditions. Detection of retrovirus HTLVII[-LAV, which is known to exist in We provide a highly sensitive method that can be used to Such a result is very This detection method is advantageous for many people, and the specialized literature also shows that such detection methods cannot be used in practice. This is surprising given that it was written that way.

In addition, research work on sondes is carried out under optimal conditions, using cultured cells in vitro. only. This is completely different from applications within ecology.

Figure 1 Figure 2 10 days Figure 3 international search report INTER,’JATrONAL APPLICATION No, PCT/ E’R8710022B (SA 176413)

Claims (1)

[Claims] 1. It is characterized by applying a general molecular hybridization technique including the following steps. Leto How to track the ADN sequence of lovirus HTLVIII-LAV in seropositive individuals . a) Collect blood from the subject and separate white blood cells from it using a known method to obtain lymphocytes A. Step to get DN. In addition, this lymphocyte ADN is contained in a unit amount of at least 30 μg. used. b) restricting such amount of lymphocyte ADN without nicks within the viral ADN sequence; The stage of processing by enzymes. c) The medium of step b) containing the virus in free or integrated form with a surface area of 1 c Passed through a nitrocellulose filter that can capture up to 80μ8 ADN per m2. stage. d) The product remaining on the filter is cloned into HTLVIII-LAV virus. making contact with one sonde, the sonde being marked with a gas. In addition, The sonde uses the ADN of the virus, which does not have the encryption part for the virus envelope region. It is a subcloned fragment, and the marking is ADN1 of the sonde. Activity as close as possible to the value of 5108 cpm per μg, especially 1 μg of ADN It is possible to give an activity of 5.108 to 2.109 cpm per portion. e) reading the presence of the characteristic horizontal stripes of the virus by known methods. 2. A patent characterized in that the restriction enzyme is XbaI or an enzyme equivalent thereto A method according to claim 1. 3. The unit amount of the lymphoid ADN is between about 30 μg and 50 μg. The method according to claim 1 or 2, characterized in that: 4. Using a sonde corresponding to the subclone λBH-5 from which the phage λ was excised Claims 1 to 3, characterized in that: Method. 5. The sonde is characterized by being a low temperature marker or a radioactive marker. A method according to any one of claims 1 to 4. 6. Claim 1, characterized in that it includes the following main steps: to the method described in any of paragraphs 5 to 5. (1) Phenol-chloroform after proteolysis by protein hydrolase K Extraction of long chain lymphocyte ADN by mixture. (2) Weighing the obtained ADN by spectrophotometry and measuring it on an agarose minigel. Confirmation of length and degradability by nuclease test. (3) More than 5 units of restriction enzyme BbaI per μg of ADN to be degraded Solution. (4) Restriction flag on 0.8% agarose gel in transfer buffer 10x SSC Extension of ment. (5) 32PdCTp at 800 cu per mmole for 250 μ Ci Marking of the sonde by moving the nick with +dATP or 32PdCTP only . (6) 10 mg ADN of salmon semen denatured by sonication at 67°C for 48 hours /ml pH = 7.5, 50mM Tris-HCl, 50mM EDTA, in 0.1% SDS, 10x Denhardt, 5x SSC buffer. , a cross between a filter and a modified sonde. (7) Final wash with a buffer of 1×SSC and 0.1% SDS. (8) With two reinforced screens when the filter is dry and the sensitive film is present. its inclusion in the cassette. Note that autoradiography is performed at -70°C. (9) Color development of horizontal stripes photographed by autoradiography. (10) Phage degraded by HindIII as a measure of ADN size Horizontal stripes taken autoradiographically on the illumination platform using λ reading. 7. Claims 1 to 6, characterized in that they include in part the following: A kit, box or bag for carrying out the method according to any of the preceding paragraphs. - Sonde-like zoster corresponding to subclone λBH-5 from which phage λ has been excised. HTLVIII-LAV-digested by XbaI used as a control Preparation of ADN of phage λ. - Enzyme XbaI in enzyme storage buffer. - Buffer for enzymatic digestion. 8. Claim 7, characterized in that the marker is radioactive or low temperature. Kits listed in section.