RU2186388C2 - Method for quantitative detection of hepatitis c virus rna in blood serum - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: the innovation deals with the methods for detecting the quantity of viral genomes in a blood sample. The quantitative standard for evaluating the quantity of hepatitis C virus genomes is elaborated in patients' blood serum samples. It is, also, developed the method of quantitative evaluation of hepatitis C virus RNA in blood serum by using a retroviral vector containing a modified sequence of hepatitis C virus as an internal standard to be used for amplification in PCR, and a selective marker. The suggested retroviral standard is obtained from the culture of packing cells by simply gathering the cultivation medium where cells were growing. The presence of resistance gene to puromicin in a vector enables to detect the titer of standard's viral particles in experiments on infecting the cell cultures. The obtained viral particles of the standard are not pathogenic for people and animals. EFFECT: higher efficiency and accuracy of detection. 1 dwg

Description

 The invention relates to medicine, namely to methods for determining the number of viral genomes present in a blood sample. We have developed a quantitative standard for estimating the number of hepatitis C genomes in a patient’s blood serum sample.

 Today, competitive PCR using a standard is used to quantify the content of viral nucleic acids. The internal DNA or RNA standard is amplified with the same primers as the target DNA or RNA, but includes either a deletion or an insert, so that the products obtained by amplification of the standard and the target are different. Varying known amounts of the internal standard are added to equal aliquots of the sample containing unknown amounts of the target. The internal standard and the target compete equally for binding to primers and amplification in PCR. The application efficiency in this case will be the same for two matrices. Equal amounts of products will be amplified when the initial matrix concentrations are equal. The ratio of the products obtained during the PCR process can be determined experimentally and the equivalent point can be calculated.

 The most successful in our opinion modification of the method for measuring the amount of viral nucleic acids using mutant viruses as the internal standard is described by Natarajan; Venkatachala, Germantown, MD Salzman; Norman P, Potomas, MD. Patent US 77661.

 The method proposed by Natarajan; Venkatachala, Germantown, MD Salzman; Norman P, Potomas, MD.

 The authors proposed a method for measuring viral nucleic acids in any sample using an infectious mutant virus, which has a sequence tag in the genome region used for amplification, as an internal control. Dilutions of this virus are added to equal aliquots of the sample for measurement. Nucleic acids are secreted, amplified and quantified. This method allows you to avoid errors that occur during the allocation of nucleic acids.

 In our invention, in contrast to the prototype, the internal standard is constructed on the basis of a retroviral vector into which a modified portion of the hepatitis C virus genome is cloned, used for amplification, which gives us the following advantages: the presence of the puromycin resistance gene in the vector allows us to determine the titer of viral particles of the standard in cell culture infection experiments; the preparation of a defective virus that is not capable of replication is obtained from the culture of packing cells, simply collecting the culture medium in which the cells grew; the resulting viral particles of the standard are not pathogenic to humans and animals.

 Description of the proposed method for the quantitative determination of hepatitis C virus RNA in the blood serum of patients.

 The construction of an internal standard.

The sequence containing the modified hepatitis C virus cDNA fragment (1100 bp) used for amplification in PCR with the same primers as the original hepatitis C virus fragment is inserted into the hBabe retroviral vector hRabe EcoRl site. The modified hepatitis C virus cDNA fragment has insert 628 n.p. in position 340 N.R. (Krnl). The resulting construct (DNA matrix) was transfected into the mouse packaging line PG 13 by electroporation. The PG 13 cell line was cultured in DMEM with 10% FBS. After selection on a medium containing 4 μg / ml puromycin, cloning was carried out by the method of limiting dilutions and a clone was selected. The culture medium of this clone containing the RNA matrix packed into viral particles was collected, aliquoted, frozen at -70 ^° C and used for the reverse transcription reaction and PCR.

 Primers.

For reverse transcription and PCR, primers were used (2):
SU1 5 '(GTG CTT GCG AGT GC 3'
ASU1 5 '(AGG GGTR TCG ATG AC 3'
SU2 5 '(GGA GGT CTC GTA GAC CGT G 3'
AS1b 5 '(GAG CCA TCC TGC CCA CCC CA 3'
AS2a 5'CCA AGA GGG ACG GGA FCC TC 3 '
AS1a 5 '(GGG ATA GGC TGA CGT CTA CC 3'
AS3a 5 '(ACC GTT CAG AAG TTT TAC GC 3'
(G / A) = R
RNA isolation
Blood serum containing hepatitis C virus is stored at -70 ^° C. Aliquots (25 μl) of serial dilutions of the supernatant were added to 25 μl of blood serum and lysed with 450 μl of a solution of 5.5 M guanidine isocyanate, 0.3 M sodium acetate, v / about phenol saturated with 0.1 M Tris. PH 6.5B transport RNA, 25 μg / ml. RNA was precipitated by the addition of an equal volume of isopropanol. After centrifugation, the precipitate was washed with 70% ethanol and dissolved in 25 μl of deionized water.

Reverse transcription
Collect the working mixture and incubate for 1 hour at 42 ^o C.

5 μl of RNA solution
5 μl 5 • RT buffer (250 mMTris, pH 8.3, 375 mM KCl, 15 mM MgCl ₂ )
2.5 μl 2 mM dNTR
2.5 μl 1 μM primer ASU1
2.5 μl 50 mM DTT
5 units RNAse inhibitor
10 units M-MLV reverse transcriptase
deionized water up to 25 μl.

 PCR

 A working mixture is prepared to supply a two-stage polymerase chain reaction.

 Stage 1.

2.5 μl 10 • buffer (100 mMTris, pH 8.3, 50 mM KCl, 20 mM MgCl ₂ , 40% formamide
2.5 μl 2 mM dNTR
2.5 μl 1 μM primer SU1, ASU1
5 units TAQ polymerase
deionized water up to 25 μl.

Stage 1 of the amplification reaction is carried out in a programmable thermostat under the following temperature conditions:
94 ^o C - 30 s
42 ^o C - 60 s
72 ^o C - 45 s
25 cycles
2 stage.

2.5 μl 10 • buffer (100 mMTris, pH 8.3, 50 mM KCl, 20 mM MgCl ₂ , 40% formamide
2.5 μl 2 mM dNTR
2.5 μl 1 μM primer SU2, AS1a, AS1b, AS2a, AS3a
5 units TAQ polymerase
deionized water up to 25 μl.

Stage 2 of the amplification reaction is carried out in a programmable thermostat at the following temperature conditions:
94 ^o C - 30 s
58 ^o C - 60 s
72 ^o C - 45 s
25 cycles
The analysis of PCR products was carried out using agarose gel electrophoresis containing ethidium bromide.

 Determination of titer of viral particles.

 The advantages of the proposed internal standard include the possibility of assessing the titer of viral particles in the supernatant by the number of colonies grown after infection of the cell culture on selective medium, since the pBabe retroviral vector provides transformed cells with resistance to puromycin.

 A retroviral preparation is obtained from a culture of packaging cells by simply collecting the culture medium in which the cells grew. Cell debris is removed by centrifugation. Infected cells release viral particles most actively in the exponential growth phase. The maximum virus content in the medium is achieved at the stage immediately preceding the formation of a fused monolayer. When the monolayer has formed 80-90%, the cells are placed in fresh medium and incubated for another 12-24 hours, after which the virus is collected.

 Infection of target cells.

 1. Cells are seeded in an amount of 400,000 per: cm-cup one day before infection. The number of dishes is determined by the number of suspected dilutions of the viral preparation.

 2. The culture medium is removed from the dishes and 2 ml of fresh medium containing a certain amount of virus and 8 μg / ml polybrene is poured.

 3. The cup is placed in an incubator for 2 hours.

4. The medium containing polybrene is removed from the cups and 5 ml of fresh medium is poured. Cells are left at 37 ^o C before sieving for 24 hours.

 5. Cells are placed in a medium containing puromycin.

 6. After 7-10 days of selection, the number of puromycin-resistant colonies resulting from infection is counted. The titer of the drug is defined as the number of colony forming units in 1 ml of the viral suspension.

 The titer of viral particles obtained by this method in the supernant corresponded to 1,000,000 / ml.

 An example of determining the amount of hepatitis C virus RNA in the patient's serum is shown in the drawing.

 The amplification products of the hepatitis C virus RNA fragment and internal standard are 315 n.p. and 943 n.p. respectively. Lane 2: the amounts of amplification products of the two matrices are equal (see drawing). Based on this, the amount of hepatitis C virus RNA in the serum of this patient is 20,000 copies / ml.

List of references
1. Natarajan; Venkatachala, Germantown, MD Salzman, Norman P. Potomas, MD.

 Internflly controlled virion nucleic acid amplification reaction for quantitation of virion nucleic acid. US Patent US 6,077,661 (2000).

 2. Sudarikov A. B., Ogrel OD, Method for detecting hepatitis C virus in human serum. RF patent 2150505 (2000).

Claims (1)

 A method for estimating the amount of hepatitis C virus RNA in blood serum by PCR using an internal standard, characterized in that retroviral particles obtained on the basis of the pBabe retroviral vector were used as the internal standard, the sequence containing the modified hepatitis C virus cDNA fragment was inserted into its EcoRl site (1100 n.p.) having an insert of 628 n. item in position 340 N. p. (Knl), after which the obtained DNA matrix was transfected by electroporation into the PG13 mouse packaging line and cultured, then a clone was selected in a medium containing puromycin, cultured, a culture medium containing an RNA matrix packed in viral particles was collected and used to calculate the titer of standard virus particles after infection of susceptible target cells with serial dilutions of the virus.

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