CN111004866A - Processing method of hepatitis C virus nucleic acid detection sample - Google Patents
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Abstract
The invention discloses a processing method of a hepatitis C virus nucleic acid detection sample, belonging to the technical field of in vitro detection, and the method comprises the following steps: s1, dividing the serum/plasma sample into four types, namely A jaundice, B hemolysis, C lipemia and D normal; s2, respectively centrifuging at 12000-15000 rpm for 5-10 min for three samples of A jaundice, B hemolysis and C lipemia, and not processing normal samples; respectively sucking supernate from S3, A jaundice and B hemolysis samples to obtain processed serum/plasma samples; and C, sucking the upper suspension in the lipemia sample, and sucking the serum in the middle position to obtain a treated serum/plasma sample. According to the invention, the classified samples are subjected to high-speed centrifugation, so that compared with untreated samples, experimental inhibition of the samples due to jaundice, hemolysis and lipemia is avoided, the success rate of the experiment is ensured, the generation of false negative results is avoided, and the accuracy of the detection result is improved.
Description
Technical Field
The invention relates to the technical field of in-vitro detection, in particular to a processing method of a hepatitis C virus nucleic acid detection sample.
Background
Hepatitis c is a disease that is mainly transmitted through blood, and according to the statistics of the world health organization, the global infection rate of HCV is about 3%, and about 1.8 million people are estimated to be infected with HCV, and about 3.5 ten thousand cases of new hepatitis c are generated every year. Chronic infection of Hepatitis C virus (Hepatitis C Vrius) can cause chronic inflammation, necrosis and fibrosis of liver, and some patients develop cirrhosis and even hepatocellular carcinoma (HCC), which is a serious social and public health problem due to great harm to the health and life of the patients. Therefore, the development of hepatitis C virus nucleic acid detection can well assist in hepatitis C treatment.
At present, a kit is mainly adopted for detecting hepatitis C virus, such as a hepatitis C virus nucleic acid detection kit produced by Hunan Shengxiang Biotechnology Co., Ltd, a sample needs to be pretreated before detection, and the main process is as follows: placing a pipe → a mark number → adding a reagent and a sample → mixing and standing for 30min → 1 st instant centrifugation 5s magnetic frame → abandoning the supernatant → adding HCV RNA lotion → 2 nd instant centrifugation 5s magnetic frame → abandoning the supernatant → abandoning the raffinate → adding the template and installing the machine. The result analysis is that the result of the sample is directly obtained by ABI7500 real-time fluorescence quantitative PCR instrument result calculation analysis software, if the VIC fluorescence channel amplification curve of the sample is in an obvious S type, the sample experiment is successful, and the corresponding determination result of the FAM fluorescence channel is reported; if the VIC fluorescence channel amplification curve has no value or Ct is greater than 38, the detection result of the sample is invalid.
However, the blood sample is not a perfect health condition, when the serum sample is in jaundice, hemolysis, lipemia and the like, the special sample is affected by red blood cells, bilirubin, fat particles and the like during detection, so that the HCVRNA extraction is inhibited, the result is false negative, the experiment fails in a light case, the patient needs to re-sample, the personnel and reagent cost are wasted, the patient sampling pain is increased, the result misjudgment is caused in a heavy case, and the optimal treatment time of the patient is delayed.
Disclosure of Invention
In view of the above, the present invention provides a method for processing a hepatitis c virus nucleic acid detection sample, which reduces the influence of erythrocytes, bilirubin, and fat particles on the hepatitis c virus nucleic acid detection result, and improves the success rate and accuracy of the experiment.
In order to achieve the above purpose, the invention provides the following technical scheme:
a processing method of a hepatitis C virus nucleic acid detection sample comprises the following steps:
s1, dividing the serum/plasma sample into four types, namely A jaundice, B hemolysis, C lipemia and D normal;
s2, respectively centrifuging at 12000-15000 rpm for 5-10 min for three samples of A jaundice, B hemolysis and C lipemia, and not processing normal samples;
respectively sucking supernate from S3, A jaundice and B hemolysis samples to obtain processed serum/plasma samples; and C, sucking the upper suspension in the lipemia sample, and sucking the serum in the middle position to obtain a treated serum/plasma sample.
Preferably, the processed serum/plasma sample is shaken or centrifuged for 2S to 3S before being tested on a PCR template.
Preferably, the method for processing the hepatitis c virus nucleic acid test sample comprises the following steps:
s1, dividing the serum/plasma sample into four types, namely A jaundice, B hemolysis, C lipemia and D normal;
s2, respectively centrifuging at 12000-15000 rpm for 5-10 min for three samples of A jaundice, B hemolysis and C lipemia, and not processing normal samples;
respectively sucking and discarding bottom sediments of S3, A jaundice samples and B hemolysis samples, sucking and discarding upper suspended matters of C lipemia samples, and obtaining treated serum/plasma samples;
s4, mixing the sample and the extraction reagent uniformly, and standing;
s5, placing on a magnetic separator after instantaneous centrifugation, and discarding the supernatant;
s6, adding washing liquid, shaking, mixing uniformly, performing instantaneous centrifugation, placing on a magnetic separator, and removing supernatant;
s7, placing the centrifuge tube on a magnetic separator after instantaneous centrifugation, inserting a suction head into the bottom of the centrifuge tube, and slowly and completely sucking out the liquid from the bottom and discarding the liquid;
and S8, standing, and completely sucking out and discarding the residual liquid at the bottom of the tube.
Preferably, the extraction reagent adopts an extraction reagent 1 and an extraction reagent 2, the main components of the extraction reagent 1 are sodium dodecyl sulfate, triton X-100, guanidine isothiocyanate and magnetic beads, and the main components of the extraction reagent 2 are 4-hydroxyethyl piperazine ethanesulfonic acid and sodium chloride.
The lotion adopts an extraction reagent 3 and an extraction reagent 4, the main components of the extraction reagent 3 are triton X-100 and sodium chloride, and the main component of the extraction reagent 4 is mineral oil.
Preferably, in the method for processing the hepatitis c virus nucleic acid detection sample, the method for classifying the serum sample in step S1 is:
A. jaundice sample: slightly yellow compared with normal serum/plasma by visual inspection, reddish or blackish, obvious orange yellow performance on the tube wall of the light observation tube after shaking, and determining the sample as the jaundice sample by detecting that the total bilirubin TBiL is more than 34.2 mu mol/L by a biochemical analyzer, otherwise, determining the sample as the normal serum/plasma sample;
B. hemolysis sample: slightly reddish than normal serum/plasma, darker color and weakened light transmittance by visual inspection, and a hemolyzed sample can be judged by using a colorimetry to determine that hemoglobin Hb is more than 0.05g/L, or else, the hemolyzed sample is a normal serum/plasma sample;
C. a lipemic sample: slightly turbid or not obvious compared with normal serum/plasma by visual inspection, milky white or milky yellow, and pink partial sample, wherein the sample can be judged as a lipemia sample by using a biochemical analyzer to measure triglyceride TG >4.20mmol/L, otherwise, the sample is the normal serum/plasma sample.
Compared with the prior art, the invention has the beneficial effects that:
the invention relates to a processing method of hepatitis C virus nucleic acid detection samples, which comprises the steps of carrying out high-speed centrifugation on classified samples, leading suspended particles in a serum/plasma sample to gradually sink under the action of a gravitational field, wherein the heavier the particles are, the faster the particles sink, on the contrary, the particles with the density smaller than that of the sample float upwards, and separating substances with different sedimentation coefficients and buoyancy densities in the sample by a strong centrifugal force generated by the high-speed rotation of a rotor of a centrifugal machine. After the sample is centrifuged at high speed, bilirubin and erythrocytes are mainly deposited at the bottom of the sample tube, and fat particles are suspended in the upper layer of the sample. Through classification and high-speed centrifugation pretreatment, compared with untreated samples, the method avoids experiment inhibition of the samples due to jaundice, hemolysis and lipemia, ensures the success rate of the experiment, avoids the generation of false negative results, and improves the accuracy of the detection result.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.
FIG. 1 is a graph showing the results of all (VIC, FAM, ROX) fluorescence channel amplification curves obtained after the experiment of example 1, in which jaundice, lipemia and hemolysis samples were not processed and loaded; the abscissa of the graph is the fluorescence intensity of the original amplification reaction, and the ordinate is the number of PCR cycles.
FIG. 2 is a graph showing the result of VIC fluorescence channel amplification curve obtained after the experiment of example 1, in which the samples of jaundice, lipemia and hemolysis were not treated and were loaded; the abscissa of the graph is fluorescence intensity after software analysis, and the ordinate is the number of PCR cycles.
FIG. 3 is a graph showing the results of all (VIC, FAM, ROX) fluorescence channel amplification curves obtained after centrifugation and loading of a jaundice sample, a lipemia sample, and a hemolysis sample in example 2; the abscissa of the graph is the fluorescence intensity of the original amplification reaction, and the ordinate is the number of PCR cycles.
FIG. 4 is a graph showing the result of VIC fluorescence channel amplification curve obtained after the experiment of example 2, in which the samples of jaundice, lipemia and hemolysis were centrifuged at high speed and loaded; the abscissa of the graph is fluorescence intensity after software analysis, and the ordinate is the number of PCR cycles.
FIG. 5 shows 10 untreated samples of example 1;
FIG. 6 shows 10 samples after the classification and centrifugation treatment in example 2.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail with reference to the following embodiments. The extraction reagent 1, the extraction reagent 2, the extraction reagent 3, the extraction reagent 4, the negative control, the positive control and the quantitative reference products A-D adopted by the invention are all internal products of the hepatitis C virus nucleic acid detection kit provided by the manufacturer for Hunan Shengxiang biological technology Co.
Example 1
A sample processing method of the existing hepatitis C virus nucleic acid detection kit (Hunan Shengxiang Biotechnology Co., Ltd.) comprises the following steps:
1. taking 10 serum samples distributed into four types, wherein the samples are numbered from 1 to 10, wherein the samples 1 to 9 are visually detected as abnormal samples, and 10 is a sample (control), and the sample is shown in figure 5;
2. taking a proper amount of 1.5ml of a sterilized centrifuge tube, respectively marking a negative control, a positive control, quantitative reference products A-D and a sample to be detected, and adding 600 mul of mixed solution of the extraction reagent 1 into each tube;
3. adding 200 mul of sample to be tested, negative control, positive control and quantitative reference substances A-D into each tube, covering a tube cover, shaking and uniformly mixing for 10 seconds, and performing instantaneous centrifugation;
4. adding 100 mul of extraction reagent 2 into each tube, shaking and uniformly mixing for 10 seconds, and standing for 30 minutes at room temperature;
5. after instantaneous centrifugation, the tube was placed on a magnetic rack, and after 3 minutes the solution was slowly aspirated (taking care not to touch the brown magnetic beads-containing material adsorbed to the tube wall);
6. adding 600 mul of extraction reagent 3 and 200 mul of extraction reagent 4 into each tube, shaking and uniformly mixing for 5 seconds, and placing the centrifugal tube on a magnetic frame again after instantaneous centrifugation;
7. and after 3 minutes, inserting the suction head into the bottom of the centrifuge tube, slowly and completely sucking out and discarding the liquid from the bottom, standing for 1 minute, completely sucking out and discarding the residual liquid at the bottom of the centrifuge tube, and waiting for detection on a computer.
Example 2
The invention relates to a processing method of a hepatitis C virus nucleic acid detection sample, which comprises the following steps:
four types of serum samples were distributed in 10 samples labeled 1-10 in example 1, and the serum/plasma samples were classified into four types, i.e., jaundice, hemolysis B, lipemia C, and normal D, according to the following classification methods:
A. jaundice sample: slightly yellow compared with normal serum/plasma by visual inspection, reddish or blackish, obvious orange yellow performance on the tube wall of the light observation tube after shaking, and determining the sample as the jaundice sample by detecting that the total bilirubin TBiL is more than 34.2 mu mol/L by a biochemical analyzer, otherwise, determining the sample as the normal serum/plasma sample;
B. hemolysis sample: slightly reddish than normal serum/plasma, darker color and weakened light transmittance by visual inspection, and a hemolyzed sample can be judged by using a colorimetry to determine that hemoglobin Hb is more than 0.05g/L, or else, the hemolyzed sample is a normal serum/plasma sample;
C. a lipemic sample: slightly turbid or not obvious compared with normal serum/plasma by visual inspection, milky white or milky yellow, and pink partial sample, wherein the sample can be judged as a lipemia sample by using a biochemical analyzer to measure triglyceride TG >4.20mmol/L, otherwise, the sample is the normal serum/plasma sample.
Wherein, samples 1-4 are jaundice samples, samples 5, 6 are hemolysis samples, samples 7-9 are lipemia samples, and sample 10 is a normal sample (as a control);
2. centrifuging 10 samples at 13200rpm for 10min on an Ebende high-speed refrigerated centrifuge before the experiment, sucking supernate from jaundice samples 1-4 and hemolysis samples 5 and 6, discarding upper-layer grease from lipemia samples 7-9, sucking middle-position serum, and not processing No. 10 (control) sample as shown in FIG. 6;
3. same as example 1, steps 2-6;
4. after 3 minutes, inserting the suction head into the bottom of the centrifuge tube, slowly and completely sucking out and discarding the liquid from the bottom, and placing the centrifuge tube on the magnetic frame again after instantaneous centrifugation for 2 seconds; and standing for 1 minute, completely sucking out residual liquid at the bottom of the tube, discarding, and waiting for detection on a computer.
Example 3
The invention relates to a processing method of a hepatitis C virus nucleic acid detection sample, which comprises the following steps:
1. taking 10 distributed four types of serum samples of the labels 1-10 classified in the embodiment 2;
2. centrifuging 10 samples on an Ebende high-speed refrigerated centrifuge at 12000rpm for 10min before experiment, sucking supernate from jaundice samples 1-4 and hemolysis samples 5 and 6, discarding upper-layer grease from lipemia samples 7-9, sucking middle-position serum, and not processing No. 10 sample;
3. same as example 1, steps 2-6;
4. after 3 minutes, inserting the suction head into the bottom of the centrifuge tube, slowly and completely sucking out and discarding the liquid from the bottom, and placing the centrifuge tube on the magnetic frame again after instantaneous centrifugation for 3 seconds; and standing for 1 minute, completely sucking out residual liquid at the bottom of the tube, discarding, and waiting for detection on a computer.
Example 4
The invention relates to a processing method of a hepatitis C virus nucleic acid detection sample, which comprises the following steps:
1. taking 10 distributed four types of serum samples of the labels 1-10 classified in the embodiment 2;
2. centrifuging 10 samples at 15000rpm for 5min on an Ebende high-speed refrigerated centrifuge before experiment, sucking supernate from jaundice samples 1-4 and hemolysis samples 5 and 6, discarding upper-layer grease from lipemia samples 7-9, sucking middle-position serum, and not processing No. 10 sample;
3. same as example 1, steps 2-6;
4. after 3 minutes, inserting the suction head into the bottom of the centrifuge tube, slowly and completely sucking out and discarding the liquid from the bottom, and placing the centrifuge tube on the magnetic frame again after shaking for 2 seconds; and standing for 1 minute, completely sucking out residual liquid at the bottom of the tube, discarding, and waiting for detection on a computer.
Example 5
Taking samples treated in the example 1 and the example 2, adding 50 μ l of PCR-mix into each tube, sucking the PCR mixed liquor by a suction head to elute brown residues adsorbed on the wall of a centrifugal tube, repeating the steps for several times to completely elute the brown residues as much as possible, transferring the eluted brown mixed liquor into a 0.2ml PCR reaction tube, covering a tube cover, and transferring the tube cover to an ABI7500 real-time fluorescence quantitative PCR instrument for detection.
And (4) analyzing results: the quantitative results of the samples in the examples 1 and 2 are directly obtained by ABI7500 real-time fluorescence quantitative PCR instrument result calculation analysis software, the sample VIC fluorescence channel amplification curve of the example 1 has no numerical value (as shown in figure 1-2), the detection result is invalid, and the whole experiment fails. In example 2, the amplification curve of the VIC fluorescence channel (shown in FIGS. 3-4) appeared in the samples treated by the method of the present invention, and the experiment was successful. The curves for examples 3-4 are similar to example 2, indicating successful experimentation.
The method of the present invention is a method for pretreating a sample, and is not a method for diagnosing and treating a disease in order to obtain an intermediate result. The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: it is to be understood that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof, but such modifications or substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1. A method for processing a hepatitis C virus nucleic acid detection sample is characterized by comprising the following steps:
s1, dividing the serum/plasma sample into four types, namely A jaundice, B hemolysis, C lipemia and D normal;
s2, respectively centrifuging at 12000-15000 rpm for 5-10 min for three samples of A jaundice, B hemolysis and C lipemia, and not processing normal samples;
respectively sucking supernate from S3, A jaundice and B hemolysis samples to obtain processed serum/plasma samples; and C, sucking the upper suspension in the lipemia sample, and sucking the serum in the middle position to obtain a treated serum/plasma sample.
2. The method for processing a hepatitis C virus nucleic acid test sample according to claim 1, comprising the steps of: and (3) vibrating or centrifuging the processed serum/plasma sample for 2-3 seconds before adding a PCR template and performing on-machine detection.
3. The method for processing a hepatitis C virus nucleic acid test sample according to claim 1, comprising the steps of:
s1, dividing the serum/plasma sample into four types, namely A jaundice, B hemolysis, C lipemia and D normal;
s2, respectively centrifuging at 12000-15000 rpm for 5-10 min for three samples of A jaundice, B hemolysis and C lipemia, and not processing normal samples;
respectively sucking and discarding bottom sediments of S3, A jaundice samples and B hemolysis samples, sucking and discarding upper suspended matters of C lipemia samples, and obtaining treated serum/plasma samples;
s4, mixing the sample and the extraction reagent uniformly, and standing;
s5, placing on a magnetic separator after instantaneous centrifugation, and discarding the supernatant;
s6, adding washing liquid, shaking, mixing uniformly, performing instantaneous centrifugation, placing on a magnetic separator, and removing supernatant;
s7, placing the centrifuge tube on a magnetic separator after instantaneous centrifugation, inserting a suction head into the bottom of the centrifuge tube, and slowly and completely sucking out the liquid from the bottom and discarding the liquid;
and S8, standing, and completely sucking out and discarding the residual liquid at the bottom of the tube.
4. The method for processing the hepatitis C virus nucleic acid detection sample according to claim 3, wherein the extraction reagent comprises an extraction reagent 1 and an extraction reagent 2, the main components of the extraction reagent 1 are sodium dodecyl sulfate, triton X-100, guanidine isothiocyanate and magnetic beads, and the main components of the extraction reagent 2 are 4-hydroxyethyl piperazine ethanesulfonic acid and sodium chloride.
5. The method for processing a hepatitis C virus nucleic acid test sample according to claim 3, wherein the washing reagent comprises extraction reagent 3 and extraction reagent 4, the main components of extraction reagent 3 are Triton X-100 and sodium chloride, and the main component of extraction reagent 4 is mineral oil.
6. The method for processing a hepatitis C virus nucleic acid detection sample according to any one of claims 1 to 5, wherein the method for classifying a serum sample in step S1 is:
A. jaundice sample: slightly yellow compared with normal serum/plasma by visual inspection, reddish or blackish, obvious orange yellow performance on the tube wall of the light observation tube after shaking, and determining the sample as the jaundice sample by detecting that the total bilirubin TBiL is more than 34.2 mu mol/L by a biochemical analyzer, otherwise, determining the sample as the normal serum/plasma sample;
B. hemolysis sample: slightly reddish than normal serum/plasma, darker color and weakened light transmittance by visual inspection, and a hemolyzed sample can be judged by using a colorimetry to determine that hemoglobin Hb is more than 0.05g/L, or else, the hemolyzed sample is a normal serum/plasma sample;
C. a lipemic sample: slightly turbid or not obvious compared with normal serum/plasma by visual inspection, milky white or milky yellow, and pink partial sample, wherein the sample can be judged as a lipemia sample by using a biochemical analyzer to measure triglyceride TG >4.20mmol/L, otherwise, the sample is the normal serum/plasma sample.
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| CN102154510A (en) * | 2011-02-25 | 2011-08-17 | 湖南圣湘生物科技有限公司 | Nucleic acid quantitative detection kit for hepatitis C virus (HCV) |
| US20160349237A1 (en) * | 2014-02-19 | 2016-12-01 | Roche Diagnostics Operations, Inc. | Method and device for assigning a blood plasma sample |
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