Apparatus for quantitative analysis of a nucleic acid amplification reaction
First Claim
1. An apparatus for determining an unknown starting quantity of a target nucleic acid sequence in a test sample, the apparatus comprising:
- a) at least one detection mechanism for measuring;
i) signals indicative of the respective quantities of the target nucleic acid sequence and of a first internal control being amplified in a first nucleic acid amplification reaction, wherein the first internal control comprises a second nucleic acid sequence different than the target nucleic acid sequence;
ii) signals indicative of the respective quantities of a first standard and of a second internal control being amplified in a second nucleic acid amplification reaction, wherein the first standard comprises a first known starting quantity of a calibration nucleic acid sequence different than the second nucleic acid sequence, and wherein the second internal control comprises the second nucleic acid sequence;
iii) signals indicative of the respective quantities of at least a second standard and of a third internal control being amplified in a third nucleic acid amplification reaction, wherein the second standard comprises a second known starting quantity of the calibration nucleic acid sequence, the third internal control comprises the second nucleic acid sequence, and the starting quantity of the second nucleic acid sequence is substantially equal in each of the amplification reactions;
b) at least one controller in communication with the detection mechanism, wherein the controller is programmed to perform the steps of;
i) determining from the measured signals respective threshold values for each of the standards, each of the internal controls, and the target nucleic acid sequence in the test sample;
ii) normalizing the threshold value determined for the target nucleic acid sequence in the test sample to the threshold value determined for the first internal control;
iii) normalizing the threshold values determined for the first and second standards to the threshold values determined for the second and third internal controls, respectively;
iv) deriving a calibration curve from the known starting quantities and the normalized threshold values of the standards; and
v) determining the starting quantity of the target nucleic acid sequence in the test sample using the calibration curve and the normalized threshold value determined for the target nucleic acid sequence.
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Abstract
An apparatus for determining a threshold value (e.g., a threshold cycle number or a time value) in a nucleic acid amplification reaction comprises a detection mechanism for measuring, at a plurality of different times during the amplification reaction, at least one signal whose intensity is related to the quantity of a nucleic acid sequence being amplified in the reaction. The apparatus also includes a controller in communication with the detection mechanism. The controller is programmed to perform the steps of deriving a growth curve from the measurements of the signal; calculating a derivative of the growth curve; identifying a characteristic of the derivative; and determining a threshold value associated with the characteristic of the derivative. Embodiments of an apparatus for determining a starting quantity of a nucleic acid sequence in a test sample are also provided.
91 Citations
48 Claims
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1. An apparatus for determining an unknown starting quantity of a target nucleic acid sequence in a test sample, the apparatus comprising:
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a) at least one detection mechanism for measuring;
i) signals indicative of the respective quantities of the target nucleic acid sequence and of a first internal control being amplified in a first nucleic acid amplification reaction, wherein the first internal control comprises a second nucleic acid sequence different than the target nucleic acid sequence;
ii) signals indicative of the respective quantities of a first standard and of a second internal control being amplified in a second nucleic acid amplification reaction, wherein the first standard comprises a first known starting quantity of a calibration nucleic acid sequence different than the second nucleic acid sequence, and wherein the second internal control comprises the second nucleic acid sequence;
iii) signals indicative of the respective quantities of at least a second standard and of a third internal control being amplified in a third nucleic acid amplification reaction, wherein the second standard comprises a second known starting quantity of the calibration nucleic acid sequence, the third internal control comprises the second nucleic acid sequence, and the starting quantity of the second nucleic acid sequence is substantially equal in each of the amplification reactions;
b) at least one controller in communication with the detection mechanism, wherein the controller is programmed to perform the steps of;
i) determining from the measured signals respective threshold values for each of the standards, each of the internal controls, and the target nucleic acid sequence in the test sample;
ii) normalizing the threshold value determined for the target nucleic acid sequence in the test sample to the threshold value determined for the first internal control;
iii) normalizing the threshold values determined for the first and second standards to the threshold values determined for the second and third internal controls, respectively;
iv) deriving a calibration curve from the known starting quantities and the normalized threshold values of the standards; and
v) determining the starting quantity of the target nucleic acid sequence in the test sample using the calibration curve and the normalized threshold value determined for the target nucleic acid sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
i) deriving a growth curve from the measurements of the signals;
ii) calculating a derivative of the growth curve;
iii) identifying a characteristic of the derivative; and
iv) determining the threshold value associated with the characteristic of the derivative.
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5. The apparatus of claim 4, wherein the step of calculating a derivative of the growth curve comprises calculating a second derivative of the growth curve, and wherein the characteristic comprises a positive peak of the second derivative.
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6. The apparatus of claim 4, wherein the step of calculating a derivative of the growth curve comprises calculating a second derivative of the growth curve, and wherein the characteristic comprises a negative peak of the second derivative.
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7. The apparatus of claim 4, wherein the step of calculating a derivative of the growth curve comprises calculating a second derivative of the growth curve, and wherein the characteristic comprises a zero crossing of the second derivative.
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8. The apparatus of claim 4, wherein the step of calculating a derivative of the growth curve comprises calculating a first derivative of the growth curve, and wherein the characteristic comprises a positive peak of the first derivative.
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9. The apparatus of claim 4, wherein the step of calculating a derivative of the growth curve comprises calculating second derivative values of the growth curve at a number of different measurement points in the reaction to yield a plurality of second derivative data points, the characteristic comprises a positive peak of the second derivative, and the step of determining the threshold value associated with the positive peak comprises:
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i) fitting a second order curve to the second derivative data points; and
ii) calculating the threshold value as the location of a peak of the second order curve.
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10. The apparatus of claim 1, wherein the step of normalizing the threshold value determined for the target nucleic acid sequence in the test sample to the threshold value determined for the first internal control comprises dividing the threshold value determined for the target nucleic acid sequence by the threshold value determined for the first internal control.
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11. The apparatus of claim 1, wherein the step of normalizing the threshold values determined for the first and second standards to the threshold values determined for the second and third internal controls, respectively, comprises dividing the threshold values determined for the first and second standards by the threshold values determined for the second and third internal controls, respectively.
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21. The apparatus of claim 1, wherein the controller is programmed to determine a respective threshold value for a standard, internal control, or target nucleic acid sequence by:
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1) storing signal values defining a growth curve for the standard, internal control, or target nucleic acid sequence, wherein the growth curve expresses signal intensity as a function of cycle number or as a function of time;
2) determining a derivative of the growth curve, wherein the derivative is determined with respect to cycle number or time; and
3) calculating a cycle number or time value associated with a characteristic of the derivative.
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22. The apparatus of claim 21, wherein each of the threshold values comprises a cycle number.
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23. The apparatus of claim 21, wherein each of the threshold values comprises an elapsed time of amplification.
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24. The apparatus of claim 21, wherein the controller is programmed to perform steps (2) and (3) of claim 21 by determining the second derivative of the growth curve and calculating the cycle number or time value as the location, in cycles or in time of amplification, of a maximum of the second derivative.
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25. The apparatus of claim 21, wherein the controller is programmed to perform steps (2) and (3) of claim 21 by determining the second derivative of the growth curve and calculating the cycle number or time value as the location, in cycles or in time of amplification, of a minimum of the second derivative.
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26. The apparatus of claim 21, wherein the controller is programmed to perform steps (2) and (3) of claim 21 by determining the second derivative of the growth curve and calculating the cycle number or time value as the location, in cycles or in time of amplification, of a zero-crossing of the second derivative.
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27. The apparatus of claim 21, wherein the controller is programmed to perform steps (2) and (3) of claim 21 by determining the first derivative of the growth curve and calculating the cycle number or time value as the location, in cycles or in time of amplification, of a maximum of the first derivative.
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28. The apparatus of claim 21, wherein the characteristic of the derivative comprises a maximum of the second derivative, and wherein the controller is programmed to perform steps (2) and (3) of claim 21 by:
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calculating second derivative values of the growth curve, with respect to cycle number or time, at a number of different measurement points to yield a plurality of second derivative data points;
fitting a second curve to at least three of the second derivative data points; and
calculating the cycle number or time value as the location, in cycles or in time of amplification, of a positive peak of the second curve.
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29. The apparatus of claim 28, wherein the cycle number or time value at the peak of the second curve is calculated using ratios of determinants, and wherein the determinants are calculated using the three second derivative data points.
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30. The apparatus of claim 21, wherein the characteristic of the derivative comprises a minimum of the second derivative, and wherein the controller is programmed to perform steps (2) and (3) of claim 21 by:
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calculating second derivative values of the growth curve, with respect to cycle number or time, at a number of different measurement points to yield a plurality of second derivative data points;
fitting a second curve to at least three of the second derivative data points; and
calculating the cycle number or time value as the location, in cycles or in time of amplification, of a negative peak of the second curve.
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31. The apparatus of claim 30, wherein the cycle number or time value at the peak of the second curve is calculated using ratios of determinants, and wherein the determinants are calculated using the three second derivative data points.
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32. The apparatus of claim 21, wherein the characteristic of the derivative comprises a maximum of the first derivative, and wherein the controller is programmed to perform steps (2) and (3) of claim 21 by:
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calculating first derivative values of the growth curve, with respect to cycle number or time, at a number of different measurement points to yield a plurality of first derivative data points;
fitting a second curve to at least three of the first derivative data points; and
calculating the cycle number or time value as the location, in cycles or in time of amplification, of a peak of the second curve.
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33. The apparatus of claim 32, wherein the cycle number or time value at the peak of the second curve is calculated using ratios of determinants, and wherein the determinants are calculated using the three first derivative data points.
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34. The apparatus of claim 21, wherein the characteristic of the derivative comprises a zero-crossing of the second derivative, and wherein the controller is programmed to perform steps (2) and (3) of claim 21 by:
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calculating second derivative values of the growth curve at a number of different measurement points to yield a plurality of second derivative data points; and
calculating the cycle number or time value at the zero-crossing by interpolation between at least two of the second derivative data points.
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12. An apparatus for determining an unknown starting quantity of a first nucleic acid sequence in a test sample, the apparatus comprising:
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a) at least one detection mechanism for detecting and measuring signals indicative of the respective quantities of the first nucleic acid sequence and at least first and second internal standards being amplified in the same reaction vessel with the first nucleic acid sequence, wherein the first standard comprises a known starting quantity of a second nucleic acid sequence different than the first nucleic acid sequence, and wherein the second standard comprises a known starting quantity of a third nucleic acid sequence different than the first and second sequences;
b) at least one controller in communication with the detection mechanism, wherein the controller is programmed to perform the steps of;
i) determining from the signals respective threshold values for each of the internal standards and for the first nucleic acid sequence;
ii) deriving a calibration curve from the known starting quantities and from the threshold values determined for the internal standards; and
iii) determining the starting quantity of the first nucleic acid sequence using the calibration curve and the threshold value determined for the first nucleic acid sequence. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
i) deriving a growth curve from the measurements of the signals;
ii) calculating a derivative of the growth curve;
iii) identifying a characteristic of the derivative; and
iv) determining the threshold value associated with the characteristic of the derivative.
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16. The apparatus of claim 15, wherein the step of calculating a derivative of the growth curve comprises calculating a second derivative of the growth curve, and wherein the characteristic comprises a positive peak of the second derivative.
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17. The apparatus of claim 15, wherein the step of calculating a derivative of the growth curve comprises calculating a second derivative of the growth curve, arid wherein the characteristic comprises a negative peak of the second derivative.
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18. The apparatus of claim 15, wherein the step of calculating a derivative of the growth curve comprises calculating a second derivative of the growth curve, and wherein the characteristic comprises a zero crossing of the second derivative.
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19. The apparatus of claim 15, wherein the step of calculating a derivative of the growth curve comprises calculating a first derivative of the growth curve, and wherein the characteristic comprises a positive peak of the first derivative.
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20. The apparatus of claim 15, wherein the step of calculating a derivative of the growth curve comprises calculating second derivative values of the growth curve at a number of different measurement points in the reaction to yield a plurality of second derivative data points, the characteristic comprises a positive peak of the second derivative, and the step of determining the threshold value associated with the positive peak comprises:
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i) fitting a second order curve to the second derivative data points; and
ii) calculating the threshold value as the location of a peak of the second order curve.
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35. The apparatus of claim 12, wherein the controller is programmed to determine a respective threshold value for an internal standard or nucleic acid sequence by:
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1) storing signal values defining a growth curve for the standard or nucleic acid sequence, wherein the growth curve expresses signal intensity as a function of cycle number or as a function of time in the reaction;
2) determining a derivative of the growth curve, wherein the derivative is determined with respect to cycle number or time; and
3) calculating a cycle number or time value associated with a characteristic of the derivative.
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36. The apparatus of claim 35, wherein each of the threshold values comprises a cycle number.
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37. The apparatus of claim 35, wherein each of the threshold values comprises an elapsed time of amplification.
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38. The apparatus of claim 35, wherein the controller is programmed to perform steps (2) and (3) of claim 35 by determining the second derivative of the growth curve and calculating the cycle number or time value as the location, in cycles or in time of amplification, of a maximum of the second derivative.
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39. The apparatus of claim 35, wherein the controller is programmed to perform steps (2) and (3) of claim 35 by determining the second derivative of the growth curve and calculating the cycle number or time value as the location, in cycles or in time of amplification, of a minimum of the second derivative.
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40. The apparatus of claim 35, wherein the controller is programmed to perform steps (2) and (3) of claim 35 by determining the second derivative of the growth curve and calculating the cycle number or time value as the location, in cycles or in time of amplification, of a zero-crossing of the second derivative.
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41. The apparatus of claim 35, wherein the controller is programmed to perform steps (2) and (3) of claim 35 by determining the first derivative of the growth curve and calculating the cycle number or time value as the location, in cycles or in time of amplification, of a maximum of the first derivative.
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42. The apparatus of claim 35, wherein the characteristic of the derivative comprises a maximum of the second derivative, and wherein the controller is programmed to perform steps (2) and (3) of claim 35 by:
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calculating second derivative values of the growth curve, with respect to cycle number or time, at a number of different measurement points to yield a plurality of second derivative data points;
fitting a second curve to at least three of the second derivative data points; and
calculating the cycle number or time value as the location, in cycles or in time of amplification, of a positive peak of the second curve.
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43. The apparatus of claim 42, wherein the cycle number or time value at the peak of the second curve is calculated using ratios of determinants, and wherein the determinants are calculated using the three second derivative data points.
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44. The apparatus of claim 35, wherein the characteristic of the derivative comprises a minimum of the second derivative, and wherein the controller is programmed to perform steps (2) and (3) of claim 35 by:
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calculating second derivative values of the growth curve, with respect to cycle number or time, at a number of different measurement points to yield a plurality of second derivative data points;
fitting a second curve to at least three of the second derivative data points; and
calculating the cycle number or time value as the location, in cycles or in time of amplification, of a negative peak of the second curve.
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45. The apparatus of claim 44, wherein the cycle number or time value at the peak of the second curve is calculated using ratios of determinants, and wherein the determinants are calculated using the three second derivative data points.
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46. The apparatus of claim 35, wherein the characteristic of the derivative comprises a maximum of the first derivative, and wherein the controller is programmed to perform steps (2) and (3) of claim 35 by:
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calculating first derivative values of the growth curve, with respect to cycle number or time, at a number of different measurement points to yield a plurality of first derivative data points;
fitting a second curve to at least three of the first derivative data points; and
calculating the cycle number or time value as the location, in cycles or in time of amplification, of a peak of the second curve.
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47. The apparatus of claim 46, wherein the cycle number or time value at the peak of the second curve is calculated using ratios of determinants, and wherein the determinants are calculated using the three first derivative data points.
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48. The apparatus of claim 35, wherein the characteristic of the derivative comprises a zero-crossing of the second derivative, and wherein the controller is programmed to perform steps (2) and (3) of claim 35 by:
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calculating second derivative values of the growth curve at a number of different measurement points to yield a plurality of second derivative data points; and
calculating the cycle number or time value at the zero-crossing by interpolation between at least two of the second derivative data points.
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Specification