Method for amplification and detection of RNA and DNA sequences

• US 5,622,820 A
• Filed: 11/03/1994
• Issued: 04/22/1997
• Est. Priority Date: 03/10/1988
• Status: Expired due to Term

• Current Assignee: City of Hope
• Original Assignee: City of Hope
• Inventors: Rossi, John J.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/334,398
• Time in Patent Office: 901 Days
• Field of Search: 435/5, 435/6, 435/912
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/101 with an internal standard/control

Abstract

A method for detecting false negative and false positive results in a method of detecting and identifying specific human nucleic acid sequences contained in human nucleic acids in a human blood or tissue sample, comprising: (1) amplifying at least one portion of the specific nucleic acid sequence present in the sample and simultaneously simplifying a marker sequence; (2) transcribing the amplification products with an RNA polymerase to produce multiple RNA copies of each copy of specific nucleic acid sequence comprising the amplification product; and (3) identifying the transcription products.

Claims

1. A method which comprises:

(i) providing a sequence of an HIV-1 RNA present in a human blood sample;

(ii) providing a cellular marker sequence which is a beta actin sequence or a T-cell receptor constant region beta chain sequence or a T4 receptor sequence;

(iii) simultaneously subjecting said sequence of said HIV-1 RNA and said cellular marker nucleic acid sequence to amplification by the polymerase chain reaction to produce a polymerase chain reaction amplification product;

(iv) determining whether said polymerase chain reaction amplification product contains an amplification product of said sequence of said HIV-1 RNA; and

(v) determining whether said polymerase chain reaction amplification product contains an amplification product of said cellular marker nucleic acid sequence;

wherein the absence in said polymerase chain reaction amplification product of an amplification product of said sequence of an HIV-1 RNA and also of an amplification product of said cellular marker nucleic acid sequence indicates that said absence of said amplification product of said sequence of an HIV-1 RNA is a false negative reading.

5. The method of claim 1 or claim 3 in which at least one of the primers utilized in said polymerase chain reaction amplification of step (iii) includes a 5'"'"' recognition site for an RNA polymerase.

2. A method which comprises:

(i) providing a sequence of an HIV-1 RNA extracted from lymphocytes isolated from peripheral blood;

(ii) providing a cellular marker sequence which is a beta actin sequence or a T-cell receptor constant region beta chain sequence or a T4 receptor sequence;

(iii) simultaneously subjecting said sequence of said HIV-1 RNA and said cellular marker nucleic acid sequence to amplification by the polymerase chain reaction to produce a polymerase chain reaction amplification product;

(iv) determining whether said polymerase chain reaction amplification product contains an amplification product of said sequence of said HIV-1 RNA; and

(v) determining whether said polymerase chain reaction amplification product contains an amplification product of said cellular marker nucleic acid sequence;

wherein the absence in said polymerase chain reaction amplification product not only of an amplification product of said sequence of an HIV-1 RNA and also of an amplification product of said cellular marker nucleic acid sequence indicates that said absence of said amplification product of said sequence of an HIV-1 RNA in said polymerase chain reaction amplification product is a false negative reading.

3. A method which comprises:

(i) providing a sequence of an HIV-1 RNA present in a human blood sample;

(ii) providing a cellular marker nucleic acid sequence which is a beta actin sequence or a T-cell receptor constant region beta chain sequence or a T4 receptor sequence;

(iii) simultaneously subjecting said sequence of said HIV-1 RNA and said cellular marker nucleic acid sequence to amplification by the polymerase chain reaction to produce a polymerase chain reaction amplification product;

(iv) providing a probe complementary to said polymerase chain reaction amplification product of said HIV-1 sequence and a probe complementary to said polymerase chain reaction amplification product of said cellular marker nucleic acid sequence;

(v) subjecting said amplification products to hybridization with said probes;

(vi)determining whether the hybridization products contain said amplification products of said sequence of HIV-1 and said cellular marker sequence;

wherein the absence in said hybridization products of an amplification product of said sequence of HIV-1 RNA and also of said amplification product of said cellular marker sequence indicates that said absence of an HIV-1 RNA in said polymerase chain reaction amplification products is a false negative result.

4. A method which comprises:

(i) providing a human blood or tissue sample;

(ii) providing at least one portion of an HIV-1 RNA sequence present in said human blood or tissue sample;

(iii) providing a cellular marker nucleic acid sequence which is a beta actin sequence or a T-cell receptor constant region beta chain sequence or a T4 receptor sequence;

(iv) simultaneously subjecting said at least one portion of an HIV-1 RNA sequence present in said human blood or tissue sample and said cellular marker nucleic acid sequence to amplification by the polymerase chain reaction to produce a polymerase chain reaction amplification product;

(v) determining whether said polymerase chain reaction amplification product contains an amplification product of said at least one portion of said HIV-1 RNA sequence; and

(vi) determining whether said polymerase chain reaction amplification product contains an amplification product of said cellular marker nucleic acid sequence;

wherein the absence in said polymerase chain reaction amplification product of an amplification product of said single portion of an HIV-1 RNA sequence and also of an amplification product of said cellular marker nucleic acid sequence indicates that said absence of said amplification product of said single portion of an HIV-1 RNA sequence is a false negative result.

6. A method for detecting a false positive result in a PCR amplification and detection assay comprising:

(a) providing a first nucleic acid sequence which may contain a target sequence;

(b) providing a second nucleic acid sequence, wherein the second nucleic acid sequence is the same as the first nucleic acid sequence except that the number of nucleotides in the second nucleic acid sequence is different from the number of nucleotides in the first nucleic acid sequence;

(c) simultaneously amplifying said first and second nucleic acid sequences by PCR with the same primers to provide amplification products that differ in their numbers of nucleotides;

(d) determining whether each of said first and second nucleic acid sequences is present in said amplification product, wherein the presence of said first nucleic acid sequence and the absence of said second nucleic acid sequence indicates a false positive result.

7. The claim 6 method in which at least one primer of said pair of primers has a 5'"'"' terminal label which is a fluorophore or a radioisotope and in which step (d) is performed by determining whether said 5'"'"' terminal label is present in said amplification product.

11. The method of claim 6, in which said second nucleic acid sequence has more nucleotides than said first nucleic acid sequence.

12. The method of claim 6, in which said second nucleic acid sequence has 21 more nucleotides than said first nucleic acid sequence.

13. The method of claim 6, in which said first nucleic acid sequence is a 3 ORF HIV-1 sequence and said second nucleotide is a 5'"'"' LTR HIV-1 sequence which has more or less nucleotides than said first nucleic acid sequence.

8. A method for detecting a false positive result in a PCR amplification and detection assay comprising:

(a) obtaining HIV-1 RNA from human blood or tissue;

(b) providing a first RNA sequence which may contain a target sequence from said HIV-1 RNA obtained in step (a);

(c) providing a second RNA sequence, wherein the second RNA sequence is the same as the first RNA sequence except that the number of nucleotides in the second RNA sequence is different from the number of nucleotides in the first RNA sequence;

(d) simultaneously amplifying said first and second RNA sequences by PCR with the same primers to provide amplification products that differ in their numbers of nucleotides; and

(e) determining whether each of said first and second RNA sequences is present in said amplification product, wherein the presence of said first RNA sequence and the absence of said second RNA sequence indicate a false positive result.

14. The method of claim 8, in which said second RNA sequence has more nucleotides that said first RNA sequence.

15. The method of claim 8, wherein said second RNA sequence has 21 or more nucleotides than said first RNA sequence.

16. The method of claim 8, wherein said first RNA sequence is a 3'"'"' ORF HIV-1 sequence and said second RNA sequence is a 5'"'"' LTR HIV-1 sequence which has more or fewer nucleotides than said first RNA sequence.

9. A method for determining a false positive result in a PCR amplification and detection assay comprising:

(a) simultaneously amplifying by PCR a first HIV-1 RNA sequence and a second HIV-1 RNA sequence wherein

(i) said second HIV-1 RNA sequence contains more or fewer nucleotides than said first HIV-1 RNA sequence; and

(ii) said first and second HIV-1 RNA sequences are amplified by PCR to provide amplification products that differ in their number of nucleotides; and

(b) determining whether each of said first and second HIV-1 RNA sequence is present in said amplification product, wherein the presence of said first HIV-1 RNA sequence and the absence of said second HIV-1 RNA sequence indicates a false positive result.

10. The claim 9 method in which said first sequence of HIV-1 RNA is from the 3'"'"' ORF or 5'"'"' LTR portion of HIV-1 RNA.