RU2146372C1 - Method of assay of parvovirus b 19 - Google Patents

Abstract

FIELD: medicine, virology. SUBSTANCE: invention relates to effective and highly specific methods of assay of parvovirus B 19 in serum blood. Method is based on the use two-stage polymerase chain reaction involving preliminary heat treatment of analyzed sera at 95 C for 10 min. Method involves the use of sera not treated with enzyme proteinase K. At the first stage of reaction method involves the use pair at temperature annealing 44 C and consisting of the nucleotide sequences 5'-GACCTCCAAACCAC-3' and 5'-AATTGCTGATACACAG-3' and at the second stage - pair at temperature annealing 56 C consisting of the nucleotide sequences 5'-CAATTGTCACAGACACCAG-3' and 5'-TGCAATCCAGACAGGTAAG-3'. Two stages of reaction are carried out in a single tube. EFFECT: accelerated testing, decreased contamination of samples, decreased cost of analysis. 2 dwg

Description

 The invention relates to medicine, namely to the creation of medical diagnostics for determining the presence of pathogenic viruses in human blood. We have developed a diagnosticum for isolating B19 parovirus in blood serum based on a two-stage polymerase chain reaction.

 To date, there are 2 main methods for identifying parvovirus B19 DNA. The first of these is DNA hybridization [1, 2, 3]. However, this method is time consuming and laborious enough to screen a large number of samples. The second method is a two-stage polymerase chain reaction (nested-PCR). The most successful, in our opinion, modification of the determination of PV B19 DNA was described by Musiani M., Gallinella G. et al. [4].

 The method proposed by Musiani M., Gallinella G. et al.

1. To 50 μl of the analyzed serum add 100 μl of a lysis solution containing 50 mm KCl, 10 mm Tris-HCl (pH 8.3), 25 mm MgCl ₂ , 0.1 mg / ml gelatin, 0.45% NP -40; 0.45% Tween 20 and 0.06 mg of Proteinase K. The mixture was incubated at 56 ^° C for one hour, then heated for 10 minutes at 95 ^° C to inactivate the enzyme and centrifuged at 14,000 rpm for 15 minutes. The supernatant is used in PCR.

2. Prepare a working mixture for a two-stage formulation of the polymerase chain reaction (figures are based on one sample):
Stage I
a) Reaction buffer (Tris-HCl (pH 8.3) - 100 mM, MgCl ₂ - 15 mM, KCl - 500 mM, gelatin 0.1%), μl - 2.5
b) Primer (nucleotide sequence 5'-CTTTAGGTATAGCCAACTGG-3 ', 20 μM), μl - 2
c) Primer (nucleotide sequence 5'-ACACTGAGTTTACTAGTGGC-3 ', 20 μM), μl - 2
d) ATP, CTP, GTP, TTP mixture (concentration of each component 2 mM), μl - 2.5
d) DNA polymerase, units activity - 1
f) Supernatant of deproteinized and heated serum, μl - 5
g) Deionized water, μl - Up to 25
The first stage of the amplification reaction is carried out in a programmable thermostat at the following temperature conditions: 94 ^o C - 30 sec; 55 ^o C - 1 min; 72 ^o C - 1 min, the number of consecutive cycles - 35.

II stage
a) Reaction buffer (Tris-HCl (pH 8.3) - 100 mM, MgCl ₂ - 15 mM, KCl - 500 mM, gelatin 0.1%), μl - 2.5
b) Primer (nucleotide sequence 5'-CTTTAGGTATAGCCAACTGG-3 ', 20 μM), μl - 2
c) Primer (nucleotide sequence 5'-ACACTGAGTTTACTAGTGGC-3 ', 20 μM), μl - 2
d) ATP, CTP, GTP, TTP mixture (concentration of each component 2 mM), μl - 2.5
d) DNA polymerase, units activity - 1
f) Amplificate obtained as a result of stage I, μl - 5
g) Deionized water, μl - Up to 25
The second stage of the amplification reaction is carried out in a programmable thermostat at the following temperature conditions: 94 ^o C - 30 sec; 55 ^o C - 1 min; 72 ^o C - 1 min, the number of consecutive cycles - 35.

 The advantage of our invention is that the PV B19 DNA is determined directly in human serum without prior treatment with proteinase K and its subsequent inactivation. This speeds up and reduces the cost of setting the test, without reducing its sensitivity. In addition, the two pairs of primers that we developed are such that, due to a significant difference in the annealing temperature of the external and internal pairs, the participation of external primers in the second stage of the reaction is excluded, which makes it possible to carry out two stages in one reaction tube, and this, in turn, also reduces the likelihood of contamination of samples.

 Description of the proposed method for determining parvovirus B19.

1. To 50 μl of the patient’s blood serum add 100 μl of physiological saline containing 0.5% NP-40 detergent, and the mixture is heated at a temperature of 95 ^o C for 10 minutes, then centrifuged at 14000 rpm for 10 minutes The resulting supernatant is used in PCR.

2. Prepare a working mixture for a two-stage formulation of the polymerase chain reaction (figures are based on one sample):
Stage I
a) Reaction buffer (TRIS-HCl (pH 8.3) - 100 mM, MgCl ₂ - 25 mM, KCl - 500 mM, formamide - 40%), μl - 2.5
b) Primer (nucleotide sequence 5'-GACCTCCAAACCAC-3 ', 20 μM), μl - 2
c) Primer (nucleotide sequence 5'-AATTGCTGATACACAG-3 ', 20 μM), μl - 2
d) ATP, CTP, GTP, TTP mixture (concentration of each component 2 mM), μl - 2.5
d) DNA polymerase, units activity - 1
f) Supernatant of heated diluted serum, μl - 5
g) Deionized water, μl - Up to 25
The first stage of the amplification reaction is carried out in a programmable thermostat at the following temperature conditions: 94 ^o C - 1 min; 44 ^o C - 1 min; 72 ^o C - 1 min, the number of consecutive cycles - 20.

II stage
a) Reaction buffer (TRIS-HCl (pH 8.3) - 100 mM, MgCl ₂ - 25 mM, KCl - 500 mM, formamide - 40%), μl - 2.5
b) Primer (nucleotide sequence 5'-CAATTGTCACAGACACCAG-3 ', 20 μm), μl - 2
c) Primer (nucleotide sequence 5'-TGCAATCCAGACAGGTATA-3 ', 20 μM), μl - 2
d) ATP, CTP, GTP, TTP mixture (concentration of each component 2 mM), μl - 2.5
d) DNA polymerase, units activity - 1
f) Amplificate obtained as a result of stage I, μl - 5
g) Deionized water, μl - Up to 25
The second stage of the amplification reaction is carried out in a programmable thermostat at the following temperature conditions: 94 ^o C - 1 min; 56 ^o C - 1 min; 72 ^o C - 1 min, the number of consecutive cycles - 35.

 After a two-stage polymerase chain reaction, the product is subjected to agarose gel electrophoresis containing ethidium bromide. The presence of PV B19 DNA in the test sample is judged by the appearance of a fluorescence band in UV light on the corresponding track in the gel. The size of the amplification is 490 bases.

 Thus, our proposed method differs from the prototype in the following.

Firstly, in the proposed method, the long process of deproteinization of a serum sample with the enzyme proteinase K and its subsequent inactivation is replaced by 10-minute heating of the sample at 95 ^o C, which reduces the complexity, execution time, cost of analysis, and also eliminates the possibility of contamination of the sample at the preparation stage PCR, and therefore the possibility of obtaining a false positive reaction.

Secondly, two pairs of primers are selected so that the annealing temperature for the outer pair of primers (44 ^o C) is significantly lower than for the inner pair (56 ^o C). Such a difference in the annealing temperature allows us to exclude the participation of a pair of external primers in the second stage of the reaction and to obtain an amplification without impurity of non-specific reaction products. In addition, this makes it possible to carry out two stages in one reaction tube, which, in turn, also reduces the likelihood of contamination of samples.

 Examples of the use of the method are shown in FIG. 1 and 2.

List of references
1. Shade RO, Blundell MC, Cotmore SF, Tattersall P., Astell CR "Nucleotide Sequence and Genom Organization of Human Parvovirus B19 Isoleted from Serum of Child During Aplastic Crisis". J. Virol. 58: 921-936 (1986).

 2. McOmish F., Yap P. L., Jordan A., Hart H., Cohen B.J. and Simmonds P. "Detection of Parvovirus B19 in Donnated Blood: a Model System for Screening by Polymerase Chain Reception". J. Clin. Microbiol. 31: 323-328 (1993).

 3. Patent: WO 9112269-A 10 22-AUG-1991.

 4. Musiani M., Gallinella G. et al. "Persistent B19 Parvovirus Infection in Haemophilic HIV-1 Infected Patients", J. of Med. Virol., 46: 103-108, 1995.

 5. Frickhofen N., Young N.S. "A Rapid Method of Sample Preparation for Detection of DNA Viruses in Human Serum by PCR," J. Virol. Meth., 35: 65-72 (1991).

Claims (1)

A method for determining parovirus B 19 using a two-stage polymerase chain reaction, including heat treatment of samples of the studied sera at 95 ^o C for 10 min and directly two-stage polymerase chain reaction, characterized in that the PV B19 DNA is determined directly in human serum not treated with the enzyme proteinase K, followed by its inactivation and as primers in the first stage of the reaction, use a pair with an annealing temperature of 44 ^o С, consisting of 5'-GACCTCCAAACCAC-3 'and 5'-AATTGCTGATACACAG-3', and in the second stage, a pair of annealing temperature of 56 ^o C, consisting of 5'-CAATTGTCACAGACACCAG-3 'and 5-TGCAATCCAGACAGGTAAG-3', with two reaction steps carried out in one tube.

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