TWI646197B - Method for rapid detecting bovine tuberculosis and BCG strain - Google Patents

Abstract

The invention provides a method for rapidly detecting a bovine tuberculosis strain and a BCG strain, which comprises adding a first primer pair which can amplify a specific sequence of a tuberculosis group to a nucleic acid sample, and amplifying a specific sequence of a bovine tuberculosis group. a second primer pair and a third primer pair capable of amplifying a BCG strain-specific sequence, and simultaneously adding a first probe calibrated with the first fluorescent substance, a second probe calibrated with the second fluorescent substance, and a third The third probe of the fluorescent substance is calibrated, and an instant polymerase chain reaction is performed to report the tuberculosis group and the tuberculosis tuberculosis by using the fluorescence of the first fluorescent substance, the second fluorescent substance and the third fluorescent substance respectively. Amplification of the specific sequence of the group and the BCG strain, and whether the nucleic acid sample has nucleic acid derived from the tuberculosis group, the bovine tuberculosis group or the BCG strain can be discriminated.

Description

Method for rapidly detecting bovine tuberculosis and BCG strain

The present invention relates to a method for rapidly detecting bovine tuberculosis bacteria and BCG strains, in particular to an oligonucleic acid probe which utilizes three different fluorescent substances for real-time polymerase chain reaction (real-time polymerase chain reaction, real- Time PCR) simultaneously detects nucleic acids from tuberculosis, bovine tuberculosis, and BCG.

Mycobacterium bovis can cause tuberculosis common to humans and animals. The main host is the cattle and other livestock (such as sheep, goats, deer, etc.) or some wild animals. Bovine TB is congenitally treated to treat tuberculosis. The first-line drug, pyrazinamide, is resistant. Therefore, when pyricarboxymamine is used to treat tuberculosis patients infected with bovine tuberculosis, it can not get good results, but may delay the patient's condition.

However, it is clinically necessary to distinguish between infected tuberculosis patients from tuberculosis caused by Mycobacterium tuberculosis complex (MTBC). It must be cultured by the specimen and subjected to a number of biochemical characterizations, so it cannot be used as a routine test item.

In view of this, the inventors of the present invention intend to establish a simple and rapid laboratory method for detecting infection of bovine tuberculosis, and include a technique for further distinguishing BCG strain (M. bovisBCG) from bovine tuberculosis, application. Clinically reported tuberculosis In the case of case, it is possible to quickly identify whether it is caused by infection of bovine tuberculosis, to provide a reference for the physician's therapeutic use, and to avoid the multi-drug resistant tuberculosis patient caused by the wrong treatment, and if the test result is a BCG strain, the present invention can further identify the same. Is it a Connaught BCG strain (M.bovisBCG-Connaught) for bladder cancer treatment to clarify the notification of suspected tuberculosis cases. And can be further extended to the health monitoring of livestock animals.

The main object of the present invention is to provide a method for rapidly detecting bovine tuberculosis bacteria and BCG strains, and simultaneously reporting three tuberculosis bacteria and cattle by using three oligonucleic acid probes for different fluorescent substances in an instant polymerase chain reaction. Amplification of a specific nucleic acid of a tuberculosis group and a BCG strain to determine whether a nucleic acid sample contains nucleic acid derived from a tuberculosis group, a bovine tuberculosis group, and a BCG strain.

A secondary object of the present invention is to provide a method for rapidly detecting a bovine tuberculosis strain and a BCG strain. When a nucleic acid sample contains a nucleic acid derived from a BCG strain, the specificity of the Connaught BCG strain for urinary bladder cancer treatment can be amplified. The nucleic acid primer is additionally subjected to an instant polymerase chain reaction with the oligonucleic acid probe to which the amplified fluorescent substance can be reported to further determine whether the nucleic acid sample contains the nucleic acid from the Connaught BCG strain.

In order to achieve the above-mentioned purpose and efficacy, the present invention discloses a method for rapidly detecting bovine tuberculosis bacteria and BCG strains, and after obtaining a nucleic acid sample, a first primer pair, a second primer pair and a third The primer pair is added to the nucleic acid sample to amplify a first target sequence, a second target sequence and a third target sequence in the polymerase chain reaction, wherein the first target sequence is Mycobacterium tuberculosis complex. The specific sequence of MTBC), The second target sequence is a specific sequence of M. bovisfamily, and the third target sequence is a specific sequence of a BCG strain (M. bovisBCG), and is additionally labeled with a first fluorescent substance. a first probe, a second probe labeled with a second phosphor, and a third probe labeled with a third phosphor to report the first target in a polymerase chain reaction Amplification of the sequence, the second target sequence and the third target sequence, and finally measuring the first fluorescent substance, the second fluorescent substance and the third fluorescent substance when the polymerase chain reaction is performed By releasing the fluorescence, it is known whether the nucleic acid sample contains nucleic acid from a tuberculosis group, a bovine tuberculosis group or a BCG strain.

Wherein, the first primer pair comprises a first pre-introduction and a first inversion primer, the first pre-introduction comprises an oligonucleic acid sequence of sequence number: 1, the first inversion primer comprises a sequence number: 2 An oligonucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 3, the first probe comprising an oligonucleic acid sequence of SEQ ID NO: 4; the second primer pair comprising a second a pre-introduction and a second inversion primer, the second pre-introduction comprising an oligonucleic acid sequence of SEQ ID NO: 5, the second anti-initial comprising an oligonucleic acid sequence of SEQ ID NO: 6, the second target sequence (aggregating The enzyme chain-locking reaction product) comprises the SEQ ID NO: 7 second probe comprising the oligonucleic acid sequence of SEQ ID NO: 8; the third primer pair comprises a third pre-introduction and a third anti-introduction, the third pre- The primer comprises an oligonucleic acid sequence of SEQ ID NO: 9, the third inverted primer comprising an oligonucleic acid sequence of SEQ ID NO: 10, the third target sequence (polymerase chain reaction product) comprising the nucleic acid sequence of SEQ ID NO: 11. The third probe comprises SEQ ID NO: 12 oligonucleic acid sequence.

In addition, when the first fluorescent substance, the second fluorescent substance, and the fluorescent light emitted by the third fluorescent substance are simultaneously measured during the polymerase chain reaction, the nucleic acid sample is expressed. The nucleic acid from the BCG strain is included, and the nucleic acid sample can be taken at this time, and a fourth primer pair is added to the nucleic acid sample to amplify a fourth target sequence in another polymerase chain reaction, the fourth The target sequence is a specific sequence for the bladder cancer treatment of M. bovisBCG-Connaught, and a fourth probe labeled with a fourth fluorescent substance is added to the polymerase chain reaction. The amplification of the fourth target sequence is reported, and finally, when the polymerase chain reaction is performed, the fluorescence emitted by the fourth fluorescent substance is immediately measured, and it is known whether the nucleic acid sample contains the strain from Connorka BCG. Nucleic acid.

Wherein, the fourth primer pair comprises a fourth pre-introduction and a fourth inversion primer, the fourth pre-introduction comprises an oligonucleic acid sequence of sequence number: 13, the fourth inversion primer comprises a sequence number: 14 An oligonucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 15 comprising the oligonucleic acid sequence of SEQ ID NO: 16.

FIG. 1A is a flow chart (1) of a preferred embodiment of the present invention; FIG. 1B is a flow chart (2) of a preferred embodiment of the present invention; It is a graph of the instantaneous fluorescence measurement result (1) of another preferred embodiment of the present invention, showing the positive result of tuberculosis of non-bovine tuberculosis and BCG strain; the second B picture: Figure (2) shows the results of the real-time fluorescence measurement of another preferred embodiment of the invention, showing the result of the positive reaction of the non-Bacillus strain of Mycobacterium tuberculosis; the second C diagram: which is another preferred embodiment of the present invention. The real-time fluorescence measurement result graph (3) shows the positive reaction result of the BCG strain; the second D map: it is the instant fluorescence measurement result chart of another preferred embodiment of the present invention. (d) showing the results of the negative control group; Fig. 3 is a diagram showing the results of real-time fluorescence measurement of another preferred embodiment of the present invention, showing the results of dilution test of the nucleic acid sequence of Bovine M. tuberculosis.

In order to provide a better understanding and understanding of the features of the present invention and the efficacies of the present invention, the preferred embodiments and the detailed description are as follows: the present invention provides a rapid detection of bovine tuberculosis and The method of BCG strain is characterized in that three kinds of oligonucleic acid probes which bind different fluorescent substances are simultaneously detected in real-time PCR to detect tuberculosis, bovine tuberculosis and BCG. The specific nucleic acid, combined with the 96-well sample tray commonly used in the polymerase chain reaction machine, can detect up to 94 nucleic acid samples at a time (minus one for the positive control group and the negative control group), and the test can be completed within 2 hours after the machine is placed. It can greatly shorten the time required for the detection of tuberculosis, bovine tuberculosis and BCG, and is suitable for the detection of small samples and the screening of a large number of samples. It can be widely used in clinical case testing, public health monitoring or Epidemiological investigations and other fields. In addition, the present invention can further distinguish the Connaught BCG strain for the treatment of bladder cancer from the BCG strain, and contribute to the clarification of the remedy for the remedy.

First, please refer to the first A diagram and the first B diagram, which are the flow chart (1) and the step flow chart (2) of the first embodiment of the present invention; as shown in the first A diagram, the rapid detection of the cattle of the present invention The method of the tuberculosis type and the BCG strain comprises the steps of: step S10: obtaining a nucleic acid sample; and step S20: adding the first primer pair, the second primer pair and the third primer pair to the nucleic acid sample And adding a first probe labeled with the first fluorescent substance, a second probe labeled with the second fluorescent substance, and a third probe labeled with the third fluorescent substance to the nucleic acid sample; and step S30: The polymerase chain reaction is performed, and the fluorescence emitted from the first fluorescent substance, the second fluorescent substance, and the third fluorescent substance is immediately measured.

In step S10, a nucleic acid sample is obtained, which is extracted from a clinical sample or a culture strain.

In this embodiment, the method for extracting the nucleic acid sample from the culture strain is as follows: in a biosafety cabinet of the Biosafety Level 3 (BSL-3) laboratory, a ring of 0.5 μL (microliter) is selected. The cultured strain was weighed and placed in a 2 mL (ml) microcentrifuge tube containing 400 μL of a single concentration of Tris-EDTA buffer, and inactivated at 95 ° C for 20 minutes in a dry heat machine. The supernatant liquid was used as the nucleic acid sample.

In step S20, a first primer pair, a second primer pair and a third primer pair are added to the nucleic acid sample, and at the same time, one of the first probes is labeled with a first fluorescent substance, and a second The second probe of the fluorescent substance calibration and the third probe labeled with a third fluorescent substance are added to the nucleic acid sample, wherein the first primer pair can amplify a first target sequence, the first probe The needle can report amplification of the first target sequence, and the first target sequence is a specific sequence of the tuberculosis flora; the second primer pair can amplify a second target sequence, and the second probe can report the first Amplification of the second target sequence, wherein the second target sequence is a specific sequence of a bovine tuberculosis group (including a bovine tuberculosis strain and a BCG strain, a positive reaction result); the third primer pair is amplifiable a third target sequence, the third probe can report amplification of the third target sequence, and the third target The target sequence is the specific sequence of the BCG strain.

In this embodiment, the first primer pair comprises a first preamble and a first inversion primer, the first preamble comprises an oligonucleic acid sequence of sequence number: 1, the first inversion primer comprises a sequence number An oligonucleic acid sequence comprising: a nucleic acid sequence of SEQ ID NO: 3, the first probe comprising an oligonucleic acid sequence of SEQ ID NO: 4; the second primer pair comprising a second preamble and a a second reverse primer comprising the oligonucleic acid sequence of SEQ ID NO: 5, the second inverted primer comprising the oligonucleic acid sequence of SEQ ID NO: 6, and the second target sequence comprising the nucleic acid of SEQ ID NO: 7. a sequence, the second probe comprising an oligonucleic acid sequence of SEQ ID NO: 8; the third primer pair comprising a third pre-introduction and a third inversion primer, the third pre-introduction comprising the sequence number: 9 a nucleic acid sequence comprising the oligonucleic acid sequence of SEQ ID NO: 10, the third target sequence comprising the nucleic acid sequence of SEQ ID NO: 11, and the third probe comprising the oligonucleic acid sequence of SEQ ID NO: 12.

When the present invention is used for testing the nucleic acid sample extracted from the culture strain, a FAM dye can be used as the first fluorescent substance to detect the tuberculosis group, and a VIC dye is used as the second fluorescent substance to detect the bovine type. a tuberculosis group, and using NED dye as a third fluorescent substance to detect a BCG strain; when the present invention is used to test the nucleic acid sample extracted from a clinical sample, an NED dye can be used as the first fluorescent substance The tuberculosis group was detected, and the VIC dye was used as the second fluorescent substance to detect the bovine tuberculosis group, and the FAM dye was used as the third fluorescent substance to detect the BCG strain.

In this embodiment, the amount of the nucleic acid sample and other reagents extracted from the culture strain by the foregoing method is as shown in Table 1:

Among them, the double concentration of the real-time polymerase chain reaction reagent was Tajman®'s universal PCR master mix.

In step S30, a polymerase chain reaction is performed on the nucleic acid sample of each of the probes labeled with different fluorescent substances, and the nucleic acid sample is immediately measured in the polymerase chain reaction. The first fluorescent substance, the second fluorescent substance, and the fluorescent light emitted by the third fluorescent substance.

In this embodiment, the first probe, the second probe, and the third probe are hydrolyzed probes, and one end of the first probe, the second probe, and the third probe (5) '-end) respectively connecting the first fluorescent substance, the second fluorescent substance or the third fluorescent substance The other end (3'-end) is connected to a fluorescent inhibitor (quencher, In this embodiment, MGBNFQ) is used to absorb the fluorescence of the first fluorescent substance, the second fluorescent substance or the third fluorescent substance, and when the polymerase chain reaction of step S30 is performed, if the first The target sequence, the second target sequence or the third target sequence is present and amplified, and the first probe, the second probe or the third probe is hydrolyzed and released in the nucleic acid elongation reaction a fluorescent substance, the second fluorescent substance or the third fluorescent substance, such that the fluorescent light of the first fluorescent substance, the second fluorescent substance or the third fluorescent substance is no longer used by the fluorescent inhibiting substance Absorbed and able to be detected.

In this embodiment, the reaction temperature and cycle number of each stage of the instant polymerase chain reaction are set as shown in Table 2:

Through the implementation of the foregoing steps S10 to S30, the method for rapidly detecting the bovine tuberculosis bacteria and the BCG strain of the present invention can utilize the first probe calibrated with the first fluorescent substance and be calibrated with the second fluorescent substance. The second probe and the third probe calibrated by the third fluorescent substance simultaneously detect the first target sequence, the second target sequence and the third target sequence in a polymerase chain reaction, respectively. It is detected whether the nucleic acid sample contains a specific sequence of a tuberculosis group, a tuberculosis strain or a BCG strain.

According to the above, the detection result of the nucleic acid sample is judged as the step S30 in the first A picture. The following steps are shown: step S40: measuring the fluorescence emitted by the first fluorescent substance; step S42: measuring the fluorescence emitted by the second fluorescent substance; step S44: measuring the third fluorescent Fluorescence emitted by the light substance; step S50: the nucleic acid sample does not contain the nucleic acid from the tuberculosis group; step S52: the nucleic acid sample contains the nucleic acid from the tuberculosis group of the non-bovine tuberculosis group; step S54: the nucleic acid sample contains the non-nucleus a nucleic acid of a bovine M. tuberculosis strain of BCG; and a step S56: the nucleic acid sample contains a nucleic acid derived from a BCG strain.

The steps S40, S42, and S44 are sequentially used to determine whether the nucleic acid sample contains a nucleic acid derived from a tuberculosis group, a bovine tuberculosis group, or a BCG strain, and steps S50, S52, S54, and S56 are the results of the determination.

The measurement result of the fluorescence emitted by the first fluorescent substance, the second fluorescent substance and the third fluorescent substance in step S30 is first determined by the judgment formula of step S40, because the first fluorescent substance For calibrating the first target sequence (specific sequence of the tuberculosis), if the fluorescence emitted by the first fluorescent substance is not detected, it means that the nucleic acid sample is not included in the judgment result of step S50. The nucleic acid from the tuberculosis group, conversely, if the fluorescence emitted by the first fluorescent substance is detected, it means that the nucleic acid sample contains the nucleic acid from the tuberculosis group, and the judgment form of step S42 should be further performed. Judge.

Since the second fluorescent substance is used for calibrating the second target sequence (specific sequence of the bovine M. tuberculosis), if the fluorescence emitted by the second fluorescent substance is not detected, it means that the step S52 is As a result of the judgment, the nucleic acid sample contains nucleic acid from the tuberculosis group of the non-bovine tuberculosis group. Conversely, if the fluorescence emitted by the second fluorescent substance is detected, it indicates that the nucleic acid sample contains the bovine protein. The nucleic acid of the type tuberculosis group should be further judged by the judgment of step S44.

Since the third fluorescent substance is used for calibrating the third target sequence (the specific sequence of the BCG strain), if the fluorescence emitted by the third fluorescent substance is not detected, the result of the determination in step S54 is indicated. The nucleic acid sample contains nucleic acid from the bovine tuberculosis group of the non-Bacillus strain, and the sample can be directly determined to be Bovine M. tuberculosis. Conversely, if the fluorescence emitted by the third fluorescent substance is detected, it indicates As a result of the judgment in the step S56, the nucleic acid sample contains the nucleic acid derived from the BCG strain.

As shown in FIG. B, after determining in the step S56 that the nucleic acid sample contains the nucleic acid from the BCG strain, the present invention can further distinguish whether the nucleic acid sample contains the nucleic acid from the Connaught BCG strain by the following steps: Step S60: Adding a fourth primer pair to the nucleic acid sample, and simultaneously adding a fourth probe labeled with the fourth fluorescent substance to the nucleic acid sample; and step S70: performing a polymerase chain reaction, and immediately measuring the release of the fourth fluorescent substance Fluorescent.

In step S60, the nucleic acid sample obtained in step S10 is further taken, and a fourth primer pair and a fourth probe labeled with a fourth fluorescent substance are added to the nucleic acid sample, wherein the fourth primer pair is Amplifying a fourth target sequence, the fourth probe can report amplification of the fourth target sequence, and the fourth target sequence is specific to the Connaught BCG strain Sexual sequence.

In this embodiment, the fourth primer pair comprises a fourth preamble and a fourth inversion primer, the fourth preamble comprises an oligonucleic acid sequence of sequence number: 13, and the fourth inversion primer comprises a sequence number. An oligonucleic acid sequence of 14 comprising the nucleic acid sequence of SEQ ID NO: 15, the fourth probe comprising the oligonucleic acid sequence of SEQ ID NO: 16. A Cy5 dye can be used as the fourth fluorescent substance to detect the Connaught BCG strain.

In this embodiment, the amount of the nucleic acid sample and other reagents extracted from the culture strain by the foregoing method is as shown in Table 3:

Among them, the double concentration of the real-time polymerase chain reaction reagent also uses the Universal PCR master mix of Taqman®.

In step S70, the fourth primer pair is added to the nucleic acid sample of the fourth probe labeled with the fourth fluorescent substance, and the polymerase chain reaction is performed on the nucleic acid sample, and the measurement is performed immediately. The nucleic acid sample is fluorescently emitted by the fourth fluorescent substance in a polymerase chain reaction.

In this embodiment, the fourth probe is also a hydrolyzable probe, and the fourth fluorescent substance is connected to one end (5'-end), and the other end (3'-end) is connected to the fluorescent light. a suppressing substance (MGBNFQ is used in the present embodiment) to absorb the fluorescence of the fourth fluorescent substance, and when the polymerase chain reaction of step S70 is performed, if the fourth target sequence exists and is amplified, the fourth The probe is hydrolyzed and releases the fourth fluorescent substance during the nucleic acid elongation reaction, so that the fluorescent light of the fourth fluorescent substance is no longer absorbed by the fluorescent inhibiting substance, and can be detected.

Through the implementation of the foregoing steps S60 to S70, the method for rapidly detecting the bovine tuberculosis bacteria and the BCG strain of the present invention can detect the polymerase chain reaction by using the fourth probe labeled with the fourth fluorescent substance. A fourth target sequence to detect whether the nucleic acid sample contains a specific sequence of a Connaught BCG strain.

As described above, the determination result of the detection result of the nucleic acid sample is as shown in each step after step S70 in the first B diagram: step S80: measuring the fluorescence emitted by the fourth fluorescent substance; step S90: nucleic acid sample A nucleic acid comprising a BCG strain derived from a non-Connaught BCG strain; and a step S92: the nucleic acid sample contains a nucleic acid from a Connaught BCG strain.

Here, step S80 is a judgment formula for judging whether or not the nucleic acid sample contains a nucleic acid derived from a Connaught BCG strain, and steps S90 and S92 are the results of the judgment.

Determining, in step S80, the measurement result of the fluorescence emitted by the fourth fluorescent substance in step S70, because the fourth fluorescent substance is used to calibrate the fourth target sequence (Connaught Card If the fluorescence of the fourth fluorescent substance is not detected, it means that the nucleic acid sample contains the BCG strain from the non-Connaught BCG strain, as determined in step S90. The nucleic acid, conversely, if the fluorescence emitted by the fourth fluorescent substance is detected, it means that the nucleic acid sample contains the nucleic acid from the Connaught BCG strain as a result of the determination in step S92.

Please refer to the second A diagram, the second B diagram, the second C diagram, and the second diagram D, which are diagrams of the real-time fluorescence measurement results of the second embodiment of the present invention; The amount of change in the normalized reporter Rn, and the horizontal axis represents the number of cycles of the polymerase chain reaction to reflect the variation of the three types of fluorescence intensity with the increase in the number of cycles during the polymerase chain reaction. As a result, it can be judged whether or not the nucleic acid sample contains a specific sequence of a tuberculosis group, a tuberculosis strain or a BCG strain.

In this embodiment, a FAM dye is selected as the first fluorescent substance to report amplification of the first target sequence (specific sequence of tuberculosis, sequence number: 3), and the FAM dye can emit green fluorescence. In each of the figures, the change in the Rn of the fluorescence wavelength of the FAM dye is indicated by a solid green line; and the VIC dye is selected as the second fluorescent substance to report the second target sequence (specificity of the bovine tuberculosis) Sequence, sequence number: 7) amplification, VIC dye can emit blue fluorescence, in each figure, the solid blue line indicates the change of Rn of the fluorescence wavelength of VIC dye; and NED dyeing As the third fluorescent substance, the amplification of the third target sequence (the specific sequence of the BCG strain, SEQ ID NO: 11) is reported, and the NED dye can emit red fluorescence, which is indicated by a solid red line in each figure. The amount of change in Rn of the fluorescent wavelength of the NED dye.

In this example, the nucleic acid samples were taken from three culture strains of typical tuberculosis strain H37Rv, bovine tuberculosis and BCG strain 172, respectively, and 1 ng (ng) was taken. The nucleic acid sample is tested and the nucleic acid extracted from the non-tuberculosis group is used as a negative control group, wherein the second A picture is the test result of the tuberculosis strain H37Rv, and only the curve of the green solid line is visible in the figure, The nucleic acid sample contains the specific sequence of the tuberculosis flora, but does not contain the specific sequence of the bovine tuberculosis or BCG strain, and the test result is consistent with the tuberculosis strain H37Rv belongs to the tuberculosis group, but does not belong to the bovine tuberculosis group or The fact of BCG strain; the second B is the test result of bovine tuberculosis. Only the curve of green and blue solid lines can be seen in the figure, which shows that the nucleic acid sample contains the specific sequence of tuberculosis and bovine tuberculosis. However, it does not contain the specific sequence of BCG vaccine. The test results are consistent with the fact that M. tuberculosis belongs to tuberculosis and bovine tuberculosis, but it cannot be directly determined as bovine tuberculosis. It needs to be matched with a third fluorescent probe. The result (negative) can be confirmed that it is not BCG strain; the second C picture is the test result of BCG strain Tokyo 172, the curve of green, blue and red solid line can be seen in the figure. The nucleic acid sample contains the specific sequences of tuberculosis, bovine tuberculosis and BCG, and the test results are consistent with the fact that the BCG strain Tokyo 172 belongs to the tuberculosis group and the bovine tuberculosis group, and is itself a BCG strain; The graph D is the test result of the negative control group, and there is no curve of green, blue or red solid line in the figure, indicating that the nucleic acid sample does not contain the specific sequence of the tuberculosis group, the bovine tuberculosis group or the BCG strain.

It can be seen from the above that the present embodiment can distinguish the tuberculosis group (second A picture, second B picture and second C picture) and non-tuberculosis bacteria (second D picture) by using the fluorescent signal of FAM dye, and use VIC. The fluorescent signal of the dye distinguishes the bovine tuberculosis group (Fig. 2B and Fig. 2C) from the tuberculosis group (second A picture) of the non-bovine tuberculosis group, and finally uses the NED dye. The fluorescent signal distinguishes the BCG strain from the bovine tuberculosis group (second C map).

Please refer to the third figure, which is a graph of the instantaneous fluorescence measurement result according to the third embodiment of the present invention; the vertical axis represents the variation value of the normalized report signal Rn, and the horizontal axis represents the polymerase chain reaction reaction. The number of cycles is used to reflect the variation of the fluorescence intensity of a series of serially diluted bovine tuberculosis nucleic acid samples during the polymerase chain reaction reaction with the increase of the number of cycles, and the results can be seen that the fluorescence detection in this embodiment is known. The limit.

This embodiment is the same as the second embodiment, and the FAM dye report sequence number: 3 is the tuberculosis-specific sequence, and the solid green line indicates the Rn change value of the fluorescence wavelength of the FAM dye in the figure; The VIC dye report sequence number: 7 is a bovine tuberculosis-specific sequence, and the Rn change in the fluorescence wavelength of the VIC dye is detected by a solid blue line in the figure.

In this example, 1 ng/μL (Ng/μl) of Bovine M. tuberculosis nucleic acid was subjected to six 10-fold serial dilutions to obtain seven concentration-level nucleic acid samples at concentrations of 1 ng/μL and 100 pg/, respectively. μL (pick/microliter), 10 pg/μL, 1 pg/μL, 100 fg/μL, 10 fg/μL, and 1 fg/μL, and 1 μL of the nucleic acid sample was taken on the machine for real-time polymerase chain reaction test.

There are six green solid lines in the figure, from left to right, representing the fluorescent signal of FAM dye in the nucleic acid samples of 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg of nucleic acid, and there are six The curve of the solid blue line, from left to right, represents the fluorescent signal of the VIC dye in the nucleic acid samples of 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg of the nucleic acid, showing the nucleic acid amount 1 ng, 100 pg, 10 pg 1, 1pg, 100fg, 10fg of bovine tuberculosis nucleic acid samples can be measured FAM dye and VIC dye fluorescent signal, only 1fg of nucleic acid sample of bovine tuberculosis nucleic acid can not be measured, so the implementation can be known The limit of fluorescence detection in the example is 10 fg of nucleic acid molecules per reaction volume.

In addition, the inventors of the present invention further tested other standard strains by using the method for rapidly detecting bovine tuberculosis bacteria and BCG strains of the present invention to evaluate the specificity of the method of the present invention. The test results are shown in Table 4, and the positive results indicate the measured results. Corresponding fluorescent signal.

Among them, 34 strains of tuberculosis (including 12 strains of M. tuberculosis, 1 strain of M. africanum, 1 strain of M. microti, 3 strains of M. bovis and 17 strains of M. bovisBCG) , M. tuberculosis, M. africanum and M. microti only appear FAM dye Fluorescent positive results, but VIC dye fluorescence and NED dye fluorescence are negative, consistent with M. tuberculosis, M. africanum and M. microti belong to tuberculosis, but not bovine tuberculosis or BCG Facts; M.bovis showed positive results of FAM dye fluorescence and VIC dye fluorescence, but NED dye fluorescence was negative, consistent with M.bovis belongs to tuberculosis, itself is bovine tuberculosis, but not The fact of BCG strain; M.bovisBCG has the positive results of FAM dye fluorescence, VIC dye fluorescence and NED dye fluorescence, which is consistent with M.bovisBCG belongs to tuberculosis and bovine tuberculosis, and is itself The fact of BCG seedlings. Conversely, another 38 strains of non-tuberculous mycobacteria showed negative results in FAM dye fluorescence, VIC stain fluorescence, and NED stain fluorescence. The test results show that the method for rapidly detecting bovine tuberculosis bacteria and BCG strains of the present invention has good specificity.

The method for rapidly detecting bovine tuberculosis bacteria and BCG strains of the present invention comprises the technology for further detecting the Connaught BCG strain, and the inventors of the present invention use this method to test other standard BCG strains to evaluate their specificity, and the test results are shown in the table. As shown in the fifth, the positive result indicates that the corresponding fluorescent signal is measured.

Among them, 17 strains of tuberculosis contained 15 strains of BCG, and only Connaught BCG was tested. The test results were positive, and the remaining 14 strains of BCG and non-Bacillus tuberculosis strains (typical tuberculosis strain H37Rv and bovine tuberculosis) were negative. The test results show that the technique for detecting Connaught BCG strain in the present invention has good specificity.

In summary, the present invention provides a method for rapidly detecting bovine tuberculosis bacteria and BCG strains, which comprises adding a first primer pair which can amplify a specific sequence of a tuberculosis group to a nucleic acid sample, and amplifiable bovine tuberculosis a second primer pair of the group-specific sequence and a third primer pair of the amplifiable BCG-specific sequence, and a first probe labeled with the first fluorescent substance and a second probe labeled with the second fluorescent substance a needle and a third probe calibrated with a third fluorescent substance, and performing an instant polymerase chain reaction to report the tuberculosis group by using the fluorescence of the first fluorescent substance, the second fluorescent substance, and the third fluorescent substance, respectively The amplification of the specific sequence of the bovine tuberculosis group and the BCG strain can identify whether the nucleic acid sample has nucleic acid derived from the tuberculosis group, the bovine tuberculosis group or the BCG strain. After determining that the nucleic acid sample has the nucleic acid from the BCG strain, the present invention may additionally add a fourth primer pair of the exemplified sequence of the condensable Connaught BCG strain and a fourth probe labeled with the fourth fluorescent substance to the nucleic acid sample. And performing another instant polymerase chain reaction to report the amplification of the specific sequence of the Connaught BCG strain using the fourth fluorescent substance, and further discriminating whether the nucleic acid sample has the nucleic acid from the Connaught BCG strain.

The above is only the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and the variations, modifications, and modifications of the shapes, structures, features, and spirits described in the claims of the present invention. All should be included in the scope of the patent application of the present invention.

The invention is a novelty, progressive and available for industrial use, and should meet the requirements of the patent application stipulated in the Patent Law of China, and the invention patent application is filed according to law, and the prayer bureau will grant the patent as soon as possible. prayer.

<110> Department of Health and Welfare Disease Control Agency

<120> Method for rapid detection of bovine tuberculosis bacteria and BCG strains

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Claims (14)

A method for rapidly detecting bovine tuberculosis bacteria and BCG strains, comprising the steps of: obtaining a nucleic acid sample; adding a first primer pair, a second primer pair and a third primer pair to the nucleic acid sample to respectively amplify one a target sequence, a second target sequence, and a third target sequence, simultaneously calibrating one of the first probes with a first phosphor, one of the second probes with a second phosphor, and one a third fluorescent substance is labeled, the third probe is added to the nucleic acid sample to report amplification of the first target sequence, the second target sequence, and the third target sequence; and the polymerase chain reaction is performed, and the amount is instantaneously Detecting the first fluorescent substance, the second fluorescent substance, and the fluorescent light emitted by the third fluorescent substance; wherein the first target sequence is a specific sequence of the tuberculosis flora, and the second target sequence is a cow The specific sequence of the tuberculosis group, the third target sequence is a specific sequence of the BCG strain, the first fluorescent signal of the first fluorescent substance is equivalently measured, and the second fluorescent substance is not measured. When the second fluorescent signal indicates that the sample contains The nucleic acid from the tuberculosis group of the non-bovine tuberculosis group, when the first and second fluorescent signals are detected and the third fluorescent signal of the third fluorescent substance is not measured, it indicates that the sample contains non- The nucleic acid of the bovine tuberculosis flora of the BCG strain, when the first, second and third fluorescent signals are measured, indicates that the sample contains nucleic acid from the BCG strain. The method for rapidly detecting a bovine tuberculosis bacteria and a BCG strain according to the first aspect of the invention, wherein the first primer pair comprises a first pre-introduction and a first inversion primer, the first pre-introduction comprises SEQ ID NO: 1 oligonucleic acid sequence comprising the oligonucleic acid sequence of SEQ ID NO: 2, the first target sequence comprising the nucleic acid sequence of SEQ ID NO: 3. A method for rapidly detecting a Bovine M. tuberculosis strain and a BCG strain according to the first aspect of the invention, wherein the first probe comprises the oligonucleic acid sequence of SEQ ID NO: 4. The method for rapidly detecting a bovine tuberculosis strain and a BCG strain according to the first aspect of the invention, wherein the second primer pair comprises a second pre-introduction and a second inversion primer, the second pre-introduction comprises SEQ ID NO: 5 oligonucleic acid sequence, the second reverse primer comprises the oligonucleic acid sequence of SEQ ID NO: 6, and the second target sequence comprises the nucleic acid sequence of SEQ ID NO: 7. A method for rapidly detecting a bovine M. tuberculosis strain and a BCG strain according to claim 1, wherein the second probe comprises the oligonucleic acid sequence of SEQ ID NO: 8. Rapid detection of cattle type as described in item 1 of the patent application A method of tuberculosis and a BCG strain, wherein the third primer pair comprises a third pre-introduction and a third inversion primer, the third pre-introduction comprising the oligonucleic acid sequence of SEQ ID NO: 9, the third inversion The primer comprises an oligonucleic acid sequence of SEQ ID NO: 10, and the third target sequence comprises the nucleic acid sequence of SEQ ID NO: 11. A method for rapidly detecting a bovine M. tuberculosis strain and a BCG strain according to claim 1, wherein the third probe comprises the oligonucleic acid sequence of SEQ ID NO: 12. The method for rapidly detecting a bovine tuberculosis strain and a BCG strain according to the first aspect of the invention, wherein the first probe, the second probe and the third probe are hydrolyzable probes. The method for rapidly detecting a bovine tuberculosis bacteria and a BCG strain according to the first aspect of the invention, wherein the first fluorescent material is a FAM dye, and the second fluorescent material is a VIC dye, the third fluorescent material. The substance is selected from NED dye. The method for rapidly detecting a bovine tuberculosis bacteria and a BCG strain according to the first aspect of the patent application, wherein when the nucleic acid sample extracted from a clinical sample is used, the first fluorescent material is selected from NED dye. The second fluorescent material is a VIC dye, and the third fluorescent material is a FAM dye. The method for rapidly detecting a bovine tuberculosis bacteria and a BCG strain according to the first aspect of the patent application, wherein the first fluorescent substance is simultaneously measured when performing a polymerase chain reaction The second fluorescent substance and the fluorescent light released by the third fluorescent substance are subjected to the following steps: adding a fourth primer pair to the nucleic acid sample to amplify a fourth target sequence, and simultaneously One of the four fluorescent substance calibrations is added to the nucleic acid sample to report amplification of the fourth target sequence; and the polymerase chain reaction is performed, and the fluorescence emitted by the fourth fluorescent substance is immediately measured; Wherein, the fourth target sequence is a specific sequence of a Connaught BCG strain. The method for rapidly detecting a bovine tuberculosis bacteria and a BCG strain according to claim 11, wherein the fourth primer pair comprises a fourth pre-introduction and a fourth inversion primer, the fourth pre-introduction comprising SEQ ID NO: 13 oligonucleic acid sequence comprising the oligonucleic acid sequence of SEQ ID NO: 14 and the fourth target sequence comprising the nucleic acid sequence of SEQ ID NO: 15. A method for rapidly detecting a Bovine M. tuberculosis strain and a BCG strain according to claim 11, wherein the fourth probe comprises the oligonucleic acid sequence of SEQ ID NO: 16. The method for rapidly detecting a bovine tuberculosis bacteria and a BCG strain according to the invention of claim 11, wherein the fourth fluorescent material is a Cy5 dye.