Granted Patent B2

US 8,560,246 · App. 12/742,801 · Granted Oct 15, 2013

Abstract

Claims (41)

1. An automated method for simultaneously managing results from a real-time polymerase chain reaction (PCR) instrument for the identification of samples comprising high-titer nucleic acids, the method comprising:

(a) obtaining fluorescence data from sequence detection software (SDS) associated with a real-time PCR instrument;

(b) independently calculating, from the data obtained from the SDS associated with the real-time PCR instrument, a normalized fluorescence signal (R n ) of a sample during a baseline period of the real-time PCR, wherein the baseline period is from about cycle 3 to about cycle 15;

(c) determining whether the normalized fluorescence signal (R n ) increases by at least about 20% during the baseline period;

(d) flagging the sample as a potentially high-titer sample when the normalized fluorescence signal (R n ) increases by at least about 20% during the baseline period;

(e) comparing the SDS-calculated fluorescence data obtained from the SDS associated with the real-time PCR instrument to the independently calculated normalized fluorescence signal (R n ) of step (b) and

(f) verifying that the SDS-calculated fluorescence data are substantially accurate.

2. The method of claim 1 , further comprising:

(g) independently calculating a threshold cycle (C T ) value from the fluorescence data from the SDS associated with the real-time PCR instrument; and

(h) comparing the independently-calculated C T value to the SDS-calculated C T value to determine if there is a match.

3. The method of claim 2 , further comprising:

(i) indicating the SDS-calculated C T value as correct when there is a match; and

(j) indicating the occurrence of an error when there is not a match.

4. The method of claim 2 , wherein independently calculating a C T value comprises:

(i) calculating a normalized fluorescence signal (R n ) as a ratio of FAM/ROX;

(ii) calculating baseline fluorescence using a linear regression method;

(iii) calculating a difference signal between the normalized fluorescence signal (R n ) and the baseline fluorescence at each cycle;

(iv) calculating the slope and intercept of the difference signal between each cycle and the previous cycle; and

(v) calculating the C T value as the fractional cycle at which the difference signal crosses a threshold.

5. An automated method for simultaneously testing blood or plasma for the identification of samples comprising high-titer nucleic acids, the method comprising:

(a) analyzing a blood or plasma sample using a real-time polymerase chain reaction (PCR) instrument to provide PCR results;

(b) obtaining fluorescence data from sequence detection software (SDS) associated with a real-time PCR instrument;

(c) independently calculating, from the data obtained from the SDS associated with the real-time PCR instrument, a normalized fluorescence signal (R n ) of a sample during a baseline period, wherein the baseline period is from about cycle 3 to about cycle 15;

(d) determining whether the normalized fluorescence signal (R n ) increases by at least about 20% during the baseline period;

(e) flagging the blood or plasma sample as a potentially high-titer sample when the normalized fluorescence signal (R n ) increases by at least about 20% during the baseline period;

(f) comparing the SDS-calculated fluorescence data obtained from the SDS associated with the real-time PCR instrument to the independently calculated normalized fluorescence signal (R n ) of step (c); and

(g) verifying that the SDS-calculated fluorescence data are substantially accurate.

6. The method of claim 5 , further comprising:

(h) independently calculating a threshold cycle (C T ) value from the fluorescence data from the SDS associated with the real-time PCR instrument; and

(i) comparing the independently-calculated C T value to the SDS-calculated C T value to determine if there is a match.

7. The method of claim 6 , further comprising:

(j) indicating the SDS-calculated C T value as correct when there is a match; and

(k) indicating the occurrence of an error when there is not a match.

8. The method of claim 6 , wherein independently calculating a C T value comprises:

(i) calculating a normalized fluorescence signal (R n );

(ii) calculating a baseline fluorescence using a linear regression method;

(iii) calculating a difference signal between the normalized fluorescence signal (R n ) and baseline fluorescence at each cycle;

(iv) calculating the slope and intercept of the difference signal between each cycle and the previous cycle; and

(v) calculating the C T value as the fractional cycle at which the difference signal crosses a threshold.

9. The method of claim 1 , wherein the number of samples is greater than about 24.

10. The method of claim 5 , wherein the number of samples is greater than about 24.

Assignments (30)