WO2012117367A1

WO2012117367A1 - Probe and primer sequences for detection of hepatitis b virus by real-time pcr, pcr reaction mixture and kits thereof.

Info

Publication number: WO2012117367A1
Application number: PCT/IB2012/050968
Authority: WO
Inventors: Mulakkapurath Narayanan Manoj; Jagannath Manjula; Avinash Kundadka KUDVA; Bhaskaran Chandrasekhar NAIR; Pillarisetti Venkata Subbarao
Original assignee: Bigtec Private Limited
Priority date: 2011-03-03
Filing date: 2012-03-01
Publication date: 2012-09-07
Also published as: TW201247877A; AR088313A1

Abstract

The present disclosure relates to method of determining presence and quantification of Hepatitis B virus (HBV) nucleic acids in biological samples. The present disclosure also discloses probe and primer sequences set forth as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 for the detection of Hepatitis B Virus. Further, the disclosure provides for PCR reaction mixture and kit for said detection and optional quantification.

Description

Probe and primer sequences for detection of Hepatitis B virus by real-time PCR, PCR reaction mixture and kits thereof.

TECHNICAL FIELD

The present disclosure relates to method of determining presence and quantification of Hepatitis B virus (HBV) nucleic acids in biological samples. The present disclosure also discloses probe and primer sequences set forth as SEQ ID No.l , SEQ ID No.2 and SEQ ID No.3 for the detection of Hepatitis B Virus. Further, the disclosure provides for PCR reaction mixture and kit for said detection and optional quantification.

BACKGROUND HBV causes acute and chronic hepatitis (type B hepatitis), and in severe cases cirrhosis and hepatic carcinoma. Recent studies have shown that throughout the world number of people infected with hepatitis B virus (HBV) which is amounting to about 300 million.

PCR based assays for the direct detection of HBV nucleic acids in the blood/serum or plasma of an infected subject may provide an advantage in determining the exact viral load of an infected patient which will be useful for a physician to know the exact stage of infection. This may further help the physician to provide a proper therapy for the patient.

Quantifying the exact viral load can also help in monitoring the progress of anti-viral therapy. The currently used methods for the diagnosis of HBV is based on ELISA(Enzyme Linked immune sorbent assay) which are based on the presence of serum markers such as HbeAg, HbsAg, or anti-HBc IgM, anti-HBe, anti-HBs, or anti-HBc IgGs. Since ELISA based methods cannot give an insight into the exact viral load there is a need to look for a method which can give the quantitative measure of the viral load.

There is a need for an effective method in view of the aforementioned problems associated with known methods of detection, so that the same can be utilized for detecting HBV which can give both qualitative and quantitative measure of the viral load. STATEMENT OF DISCLOSURE

The present disclosure relates to a nucleotide sequence set forth in SEQ ID No.l or SEQ ID No. 2 or SEQ ID No 3; a PCR reaction mixture for detection and quantification of Hepatitis B Virus, said reaction mixture comprising nucleic acid amplification reagents, SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; a method of detecting and optionally quantifying Hepatitis B Virus, said method comprising acts of (a) obtaining a PCR reaction mixture comprising nucleic acid amplification reagents, SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, (b) subjecting a test sample to the obtained reaction mixture for PCR to obtain copies of target sequence followed by observing fluorescence signal for detecting the Hepatitis B Virus and (c) optionally constructing a Standard Curve from the detected signal for quantifying the Hepatitis B Virus; a kit for detecting Hepatitis B Virus, wherein the kit comprises amplification reagents or sequences selected from group consisting SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No 3 or any combination thereof, optionally along with instruction manual; and a method of assembling a kit for detection of Hepatitis B Virus, said method comprising step of combining probe set forth as SEQ ID No. 1 or primers set forth as SEQ ID No. 2 or SEQ ID No. 3 or any combination of sequences thereof along with amplification reagents, optionally along with instruction manual.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS:

The features of the present disclosure will become more fully apparent from the following description taken in conjunction with the accompanying drawings. Understanding that the drawings depict only several embodiments in accordance with the disclosure and are therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings:

Fig 1 illustrates a real time PCR profile for the HBV standards.

Fig 2 illustrates a log dilution curve for HBV standards. DETAILED DESCRIPTION OF DISCLOSURE:

The present disclosure relates to Nucleotide sequence set forth in SEQ ID No.l or SEQ ID No. 2 or SEQ ID No 3.

The present disclosure also relates to a PCR reaction mixture for detection and optional quantification of Hepatitis B Virus, said reaction mixture comprising nucleic acid amplification reagents, SEQ ID No. 1 , SEQ ID No. 2 and SEQ ID No. 3.

The present disclosure also relates to a method of detecting and optionally quantifying Hepatitis B Virus, said method comprising acts of (a) obtaining a PCR reaction mixture comprising nucleic acid amplification reagents, SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, (b) subjecting a test sample to the obtained reaction mixture for PCR to obtain copies of target sequence followed by observing fluorescence signal for detecting the Hepatitis B Virus and (c) optionally constructing a Standard Curve from the detected signal for quantifying the Hepatitis B Virus.

In an embodiment of the present disclosure, the SEQ ID No. 1 is a probe; and the SEQ ID No. 2 and the SEQ ID No.3 are sense and anti-sense primers respectively, corresponding to the probe of SEQ ID No. 1.

In another embodiment of the present disclosure, the SEQ ID No. 1 is conjugated with fluorophore at 5' end and quencher which can be an internal quencher within the probe or at the 3' end. In yet another embodiment of the present disclosure, the fluorophore is selected from group comprising fluorescein and fluorescein derivatives FAM, VIC, JOE, 5-(2'- aminoethyl) aminonaphthalene- 1 -sulphonic acid, coumarin and coumarin derivatives, lucifer yellow, texas red, tetramethylrhodamine, 6-Carboxy Fluorescein, tetrachloro-6- carboxyfluoroscein, 5-carboxyrhodamine and cyanine dyes, preferably 6-Carboxy Fluorescein; and the quencher is selected from a group comprising Tetra Methyl Rhodamine [TAMRA], 4'-(4-dimethylaminophenylazo) benzoic acid, 4- dimethylaminophenylazophenyl-4'-maleimide, tetramethyl rhodamine, carboxytetra methyl rhodamine and BHQ dyes, preferably Black hole quencher 1 [BHQ1].

In still another embodiment of the present disclosure, the test sample is selected from a group comprising blood, serum, and plasma or any combination thereof. In still another embodiment of the present disclosure, the fluorescence signal is generated by the cleavage of the flurophore at 5' end.

The present disclosure also relates to a kit for detecting Hepatitis B Virus, wherein the kit comprises amplification reagents or sequences selected from group consisting SEQ ID No. 1 , SEQ ID No. 2 and SEQ ID No. 3 or any combination thereof, optionally along with instruction manual.

The present disclosure also relates to a method of assembling a kit for detection of Hepatitis B Virus, said method comprising step of combining probe set forth as SEQ ID No. 1 or primers set forth as SEQ ID No. 2 or SEQ ID No. 3 or any combination of sequences thereof along with amplification reagents, optionally along with instruction manual.

In an embodiment of the present disclosure, the SEQ ID No. 1 is conjugated with fluorophore at 5' end and quencher which can be an internal quencher within the probe or at the 3' end.

In another embodiment of the present disclosure, wherein the kit also comprises of a test samples, which may include infected samples for detection andoptional quantification. The samples may also include positive or negative controls or both.

In another embodiment of the present disclosure, wherein the amplification reagents are selected from a group comprising magnesium chlorideTaq polymerase and buffer or any combination thereof. List of biological sequences of the disclosure In another embodiment of the present disclosure, wherein the probe set forth as SEQ ID No. 1 and the corresponding primers set forth as SEQ ID No 2 and SEQ ID No 3 have sequences as shown in Table 1 below:

Table - 1

In another embodiment of the present disclosure, wherein the designed Oligonucleotide' probe can be used for the detection of HBV nucleic acids in an infected sample by employing Real time PCR. The mode of detection is by measuring the increase in fluorescence during PCR.

In another embodiment of the present disclosure, the "Oligonucleotide" probe of SEQ ID No. 1 is having detectable label with fluorophore at 5' end and quencher which can be an internal quencher (within the probe) or at the 3' end. In another embodiment of the present disclosure, the fluorophore is selected from a group comprising fluorescein and fluorescein derivatives FAM, VIC, JOE, 5-(2'-aminoethyl)aminonaphthalene-l-sulphonic acid, coumarin and coumarin derivatives, lucifer yellow, texas red, tetramethylrhodamine, 6-Carboxy Fluorescein, tetrachloro-6-carboxyfluoroscein, 5- carboxyrhodamine and cyanine dyes.

In another embodiment of the present disclosure said quencher is selected from a group comprising Tetra Methyl Rhodamine, 4'-(4-dimethylaminophenylazo) benzoic acid, 4- dimethylaminophenyl-4'-maleimide, tetremethylrhodamine, carboxytetramethylrhodamine and BHQ dyes. The said fluorophore is preferably 6- Carboxy Fluorescein [FAM] and the quencher is Black hole quencher 1 [BHQl] when present at the 3' end or within the probe. The present disclosure is also in relation to a method for detecting Hepatitis B Virus, where in the said PCR mixture comprising of nucleic acid amplification reagents, "Oligonucleotide" probe designated as SEQ ID No. 1 , along with its corresponding primers of SEQ ID Nos. 2 & 3 and a test sample is subjected for amplification using real- time PCR to obtain copies of the target sequence. The amplification is measured in terms of increase in fluorescence signal.

In another embodiment of the present disclosure, wherein the "Oligonucleotide" probe has a size of 21 nucleotides and the primers have a size of 16 and 24 nucleotides respectively. The designed probe has a fluorophore at the 5'end and a quencher at the 3' end or within the probe.

In another embodiment of the present disclosure, wherein the said fluorophore is preferably 6-Carboxy Fluorescein [FAM] and the quencher is Black hole quencher 1. The current disclosure is used for the detection of hepatitis B virus present in blood/serum/plasma samples. The method used for detection is by monitoring the increase in fluorescence during the PCR.

According to the present disclosure the Oligonucleotide" probe refers to a short sequence of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The "Oligonucleotide" probes can specifically hybridise to nucleic acids from all hepatitis B virus (HBV) genotypes. The "Oligonucleotide" probe according to the present disclosure is generally of 21 nucleotides length. The "Oligonucleotide" probe mentioned here specifically hybridizes to the HBV nucleic acid sequence without exhibiting non-specific hybridization to non-HBV nucleic acids.

In another embodiment of the present disclosure, wherein the "Oligonucleotide" probe employed here follows the principles of Taqman chemistry. TaqMan probes also called Double-Dye oligonucleotide or dual labeled probes, are the most widely used type of probes. They were developed by Roche [Basel, Switzerland] and ABI [Foster City, USA] from an assay that originally used a radiolabeled probe [Holland et al. 1991] and consist of a single -stranded probe sequence that is complementary to one of the strands of the amplicon. The fluorophore when excited passes its energy, via FRET (Fluorescence resonance energy transfer), to the quencher. During real time PCR the probe binds to the amplicon during each annealing step of the PCR. When the Taq polymerase extends from the primer bound to the amplicon it displaces the 5' end of the probe, which is then degraded by the 5'-3' exonuclease activity of the Taq polymerase. Cleavage continues until the remaining probe melts off the amplicon. This process releases the fluorophore and quencher into solution, specially separating them compared to when they were held together by the probe. This leads to an irreversible increase in fluorescence from the fluorophore. In another embodiment of the present disclosure, wherein the "Oligonucleotide" probe according to the present disclosure, therefore, is further provided in combination with their corresponding sense and antisense primers that can be used to specifically amplify and detect HBV nucleic acid sequences in a test sample by real time PCR.

The technology of the instant Application is further elaborated with the help of following examples. However, the examples should not be construed to limit the scope of the disclosure.

Example: 1

DNA was isolated from 10 HBV positive and 10 HBV negative serum samples using a commercial Qia amp whole blood DNA extraction kit. The extraction was done according to the manufacturer's instructions. The extraction protocol consisted of lysis, binding, washing and elution steps. The isolated DNA was subjected to real time PCR using the oligonucleotide probe designated as SEQ ID No. 1 along with its corresponding primers of SEQ ID Nos. 2 and 3 respectively. The sensitivity of the oligonucleotide probe in picking up the infected samples was compared with a commercial standard kit. Same concentrations of Real time-PCR reagents, template and primers were used in each case and also cycling conditions were kept constant for all the reactions. The composition of the real time PCR mix and PCR conditions as given in Table.2 & Table 3. Table. 2: Real time-PCR with Takara Premix

Table 3: Real time-PCR cycle conditions

Step 2 and 3 will be repeated 40 times.

Results obtained showed that the oligonucleotide probe designated as SEQ ID No. 1 picked up all the 10 positive samples within 40 cycles (positive sample cutoff) showing 100% specificity. Out of the 10 positives all the 10 were detected earlier than the commercial standard kit (Table .4). with commercial kit

None of the negative samples showed false positive with the designed oligonucleotide probe designated as SEQ ID No. 1.

Oligonucleotide of SEQ ID No.l , which is designed for the core/pre-core region of HBV genome, showed good specificity and sensitivity (100%). Out of 10 positive samples all the 10 were picked earlier than the commercial standard kit.

Example: 2

HBV DNA was isolated from a clinical sample panel of 50 serum samples using a commercial Qia amp whole blood DNA extraction kit. The isolated HBV DNA was subjected to real time-PCR using the oligonucleotide probe designated as SEQ ID No. 1 along with its corresponding primers of SEQ ID Nos. 2 and 3 respectively. The sensitivity of these oligonucleotide probes in detecting the infected samples was compared with a commercial standard Sacace PCR kit. Same concentrations of real time- PCR reagents and template were used in each case and also the cycling conditions were kept constant for all the reactions. The result obtained showed that out of 50 samples 49 samples were detected earlier than the commercial kit and only one sample that is sample 21A8 picked 0.9 Ct late than the commercial kit (Table.5). This example shows that the probe designated as SEQ ID No. 1 along with its respective primers designated as SEQ ID No. 2 and 3 to be more sensitive than the primers and probes of the commercial kit.

Table.5: Ct comparison with commercial kit

SI. No. Sample Name Ct commercial kit Ct SEQ ID No. 1

1 01A2 20.67 18.62

2 02A2 26.59 24.56

3 02A4 29.46 27.47

4 02A8 30.51 28.78

5 03A2 26.64 25.12

6 03A7 29.08 26.14

7 04A6 29.42 27.00

8 10A9 17.81 15.88

9 10D9 17.87 15.30

10 1 1 AO 27.73 26.34

11 1 1 A3 20.57 17.08

12 1 1A6 20.69 17.01

13 1 1A7 35.28 33.00

14 1 1A9 18.25 16.12

15 1 1D5 20.59 17.96

16 12 AO 29.69 28.20

17 12A4 29.30 27.22

18 12D9 22.88 18.87

19 13A2 20.39 17.94

20 20A1 20.78 17.82

21 20A5 26.23 25.85

22 21 A3 30.12 26.55 23 21A6 19.19 17.13

24 21A8 29.27 30.17

25 21A9 32.48 26.73

26 22A0 34.36 32.54

27 22A3 36.01 34.55

28 22A7 30.34 26.73

29 22D4 18.67 16.07

30 23A9 35.67 32.95

31 23D9 17.34 15.61

32 26D4 16.94 15.34

33 30A1 29.17 25.52

34 30A5 28.65 25.99

35 31A1 29.55 28.50

36 31A4 28.93 26.51

37 31A8 27.12 26.63

38 31A9 23.26 20.76

39 40A5 35.13 32.12

40 42D5 16.15 14.92

41 61D7 27.41 24.80

42 64D5 16.28 14.62

43 65D4 25.32 22.50

44 74A4 18.97 16.62

45 77A0 23.15 20.56

46 84D5 17.08 15.01

47 86D5 34.00 31.86

48 88A1 18.31 16.45

49 88A2 18.37 16.78

50 99D3 20.00 16.36 Example: 3

The efficiency of the primers and probe in detecting HBVDNA from whole blood was tested by isolating the viral DNA from the whole blood of a patient suffering from HBV infection. The isolated HBV DNA was subjected to real time-PCR using the oligonucleotide probe designated as SEQ ID No. 1 along with its corresponding primers of SEQ ID Nos. 2 and 3 respectively.

Results obtained showed that the sample was detected at 23.3 Ct. Example: 4

One can also quantify the HBV viral load by generating a log dilution curve. A log dilution curve was generated by using commercially available quantified HBV standards from Qiagen Artus kit. The HBV standards (QS 1 to QS5) were subjected to real time- PCR using the probe designated as SEQ ID No. 1 along with its corresponding primers of SEQ ID Nos. 2 and 3 and a log dilution curve was generated based upon the obtained Ct values. From the log dilution curve the copy number can be calculated in terms of IU/ml from an unknown sample (Table. 6, Fig.l & Fig.2).

Table.6: Ct obtained for HBV standards

SAMPLE

IU/ml IU/ reaction log IU/ reaction Ct

ID

QS1 1.00E+05 4.55E+05 5.658 20.59

QS2 1.00E+04 4.55E+04 4.658 23.37

QS3 1.00E+03 4.55E+03 3.658 26.66

QS4 1.00E+02 4.55E+02 2.658 30.08

QS5 l .OOE+01 4.55E+01 1.658 34.59 Based on the overall evaluation studies, SEQ ID No. 1 was considered to be the best probe for HBV detection vis-a-vis the commercial kits.

Finally the oligonucleotide probe designated as SEQ ID No. 1 can also be used for quantifying viral load in an infected sample. The primers and probes disclosed in the current disclosure are also to be provided in the form of a kit along with an instruction manual. The kit contains PCR amplification reagents such as dNTPs, Taq DNA polymerase, magnesium chloride etc along with the disclosed primers and probes. The oligonucleotide probes according to present disclosure find application for the detection of HBV infection caused by HBV.

SEQUENCE LISTING

<110> BIGTEC PRIVATE LIMITED

<120> NUCLEOTIDE SEQUENCES. REACTION MIXTURE, METHOD AND KIT THEREOF

<130> IP16525

<160> 3

<170> Patentln version 3 . 5

<210> 1

<211> 21

<212> DNA

<213> Hepatitis B virus

<220>

<221> prim_transcript

<222> (1) . . (21)

<400> 1

Tcaatcgccg cgtcgcagaa g 21

<210> 2

<211> 16

<212> DNA

<213> Hepatitis B virus

<220>

<221> primer_bind

<222> (1) . . (16)

<400> 2

Ccctcgcctc gcagac 16 <210> 3

<211> 24

<212> DNA <213> Hepatitis B virus

<220>

<221> primer_bind

<222> (1)..(24)

<400> 3

Aaagtttccc accttatgag tcca 24

Claims

We Claim

1) Nucleotide sequence set forth as SEQ ID No.l or SEQ ID No. 2 or SEQ ID No 3.

2) A PCR reaction mixture for detection and optional quantification of Hepatitis B Virus, said reaction mixture comprising nucleic acid amplification reagents, SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.

3) A method of detecting and optionally quantifying Hepatitis B Virus, said method comprising acts of:

(a) obtaining a PCR reaction mixture comprising nucleic acid amplification reagents, SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 ;

(b) subjecting a test sample to the obtained reaction mixture for PCR to obtain copies of target sequence followed by observing fluorescence signal for detecting the Hepatitis B Virus; and

(c) optionally constructing a Standard Curve from the detected signal for quantifying the Hepatitis B Virus.

4) The nucleotide sequence as claimed in claim 1 , the PCR reaction mixture as claimed in claim 2 and the method as claimed in claim 3, wherein the SEQ ID No. 1 is a probe; and the SEQ ID No. 2 and the SEQ ID No.3 are sense and anti-sense primers respectively corresponding to the probe of SEQ ID No. 1.

5) The nucleotide sequence as claimed in claim 1 , the PCR reaction mixture as claimed in claim 2 and the method as claimed in claim 3, wherein the SEQ ID No. 1 is conjugated with fluorophore at 5' end and quencher which can be an internal quencher within the probe or at the 3' end.

6) The nucleotide sequence, the PCR reaction mixture and the method as claimed in claim 5, wherein the fluorophore is selected from group comprising fluorescein and fluorescein derivatives FAM, VIC, JOE, 5-(2'-aminoethyl) aminonaphthalene-1- sulphonic acid, coumarin and coumarin derivatives, lucifer yellow, texas red, tetramethylrhodamine, 6-Carboxy Fluorescein, tetrachloro-6-carboxyfluoroscein, 5- carboxyrhodamine and cyanine dyes, preferably 6-Carboxy Fluorescein; and the quencher is selected from group comprising Terra Methyl Rhodamine [TAMRA], 4'- (4-dimethylaminophenylazo) benzoic acid, 4-dimethylaminophenylazophenyl-4'- maleimide, tetramethyl rhodamine, carboxytetra methyl rhodamine and BHQ dyes, preferably Black hole quencher 1 [BHQ1].

7) The method as claimed in claim 3, wherein the test sample is selected from a group comprising blood, serum and plasma or any combination thereof.

8) The method as claimed in claim 3, wherein the fluorescence signal is generated by the cleavage of the flurophore at 5' end.

9) A kit for detecting Hepatitis B Virus, wherein the kit comprises amplification reagents or sequences selected from group consisting SEQ ID No. 1 , SEQ ID No. 2 and SEQ ID No. 3 or any combination thereof, optionally along with instruction manual.

10) A method of assembling a kit for detection of Hepatitis B Virus, said method comprising step of combining probe set forth as SEQ ID No. 1 or primers set forth as SEQ ID No. 2 or SEQ ID No. 3 or any combination of sequences thereof along with amplification reagents, optionally along with instruction manual.

11) The kit as claimed in claim 9 and the method of assembling as claimed in claim 10, wherein the SEQ ID No. 1 is conjugated with fluorophore at 5' end and quencher which can be an internal quencher within the probe or at the 3' end.

12) The PCR reaction mixture as claimed in claim 2, the method as claimed in claim 3, the kit as claimed in claim 9 and the method of assembling as claimed in claim 10, wherein the amplification reagents are selected from a group comprising magnesium chloride, Taq polymerase and buffer or any combination thereof.