In experiments described so far and in some subsequent sections, the lysis of E. coli cells that is required to allow primers to gain access to the target plasmid has been assumed to occur in the initial heat dénaturation step of the PCR (Mahon and Lax, 1993). Although this method has allowed a good correlation between the number of cells in a PCR and the number of pQRTOl copies measured (Section 3.2.4), there are potential drawbacks involved in not having a good understanding of the lysis process. These drawbacks are: i) that the lysis may be inefficient, compromising the sensitivity of the PCR; and ii) that the lysis efficiency may be variable, depending on the influence of certain factors. Variable lysis efficiency might be encountered after storing cells at low temperatures (for example, see Ingraham (1987)), leading to apparent sample instability. It was therefore decided to look at a variety of different methods in an attempt to optimise the lysis and to minimise any artefacts that might otherwise be seen.
Several methods for the lysis of bacterial cells present in simple aqueous systems prior to PCR have been reported in the literature. These methods (which are distinct from those
used to isolate DNA from more complex systems such as soil, see for example Leff et al (1995)) can be classified into those depending on physical, chemical or enzymic methods. Examples of physical methods include the use of repeated freeze/thaw cycles (Bej et al,
1991b; Grant et al, 1992), boiling (Josephson et al, 1993; Zwadyk et al, 1994; Madico et al, 1995), sonication (Kirk and Rowe, 1994; Sparagano et al, 1994) and mechanical disruption using glass or zirconia beads (Johns et al, 1994), the latter being used to lyse spores of Bacillus anthracis. Chemical methods are illustrated by the use of detergents (Sparagano et al, 1994; Alvarez et al, 1995; Sakallah et al, 1995) or formamide (Sparagano et al, 1994). Enzymes that have been used in lysis treatments include proteinase K (Atlas and Bej, 1990; Grant et al, 1992; Goldenberger et al, 1995) and lysozyme (Grossman and Ron, 1975). Several of the above methods are not easily classified as they may use a combination of techniques; for instance, the use of proteinase K and SDS detergent (Goldenberger et al, 1995).
Several workers have compared methods side by side in an attempt to determine which is best suited to the particular application. For example, in a study by Sparagano et al (1994) sonication, freeze/thawing and formamide treatment all produced consistent positive PCR results, whilst chemical lysis and disruption using glass beads were not successful. However, this and other studies have not used truly quantitative methods to assess the relative recoveries effected by the different methods. The aim of this series of experiments was therefore to compare a cross section of different methods using QPCR to determine which gives the optimal recovery of plasmid pQRTOl. The methods used are summarised in Table 4.1.
Method type Comments Reference
A. No lysis step Control (Mahon and Lax, 1993)
B. Boiling 1 0 minutes in boiling water bath (Josephson et al, 1993)
C. Freeze/thaw 6 x 1 min cycles between ethanol/dry ice and 50°C. (Bej et al, 1991b)
D. Sonication 6 cycles: 30 s at 8 fxm amplitude, 50 Hz, 10 sec off (Kirk and Rowe, 1994)
(Soniprep, MSE); sample cooled on ice
E. Detergent 4mM SDS, 10 minute incubation at RT. (Alvarez et al, 1995)
F. Detergent ATP releasing agent (Celsis Ltd) at 1:1, 10 minute -
incubation at RT.
G. Lysozyme 0 . 1 mg mL'^ lysozyme (Sigma), 1 hour incubation at (Sparagano et al, 1994)
Chapter 4. Adaptation o f the QPCR method for air sampling
The results o f the comparison o f lysis methods are shown in Figure 4.1. Note that no results are shown for lysis methods E (SDS treatment), F (ATP releasing agent) and G (lysozyme) since, in all these cases, the PCR was inhibited. It was therefore not possible
to determine how efficient lysis was on each o f these occasions. Sparagano et a l (1994)
have similarly found that lysozyme and SDS methods o f lysis did not provide any detectable PCR product. The idea o f trying ATP releasing agent was that this solution, which contains chlorhexidine digluconate, would be effective at releasing plasmid as it is formulated to release ATP from bacteria into solution whilst not interfering with subsequent luciferase based assay.
CD CO
oontrol 10 min boil frœ ze/th a w
lysis method
so n ica te
FIG U R E 4.1 C om parison o f recoveries o f pQR7()l from w hole cells using different lysis m ethods. F igure shows recoveries o f plasm id pQR701 relative to control value (assigned 100 %). Values show n are averages (n = 2) ± SEM. All lysis m ethods were conducted u sin g 5 X 10“+ E. coli JM 107 pQR7()l cells in in 100 flL TRS, o f w hich 2 x 10 u L was assayed by PCR.
Since boiling is a very convenient and effective method to use, it was further investigated. This was achieved by carrying out an experiment where the same cell suspension was subjected to boiling for different lengths o f time. Results are shown in Figure 4.2. It is apparent from this figure that lysis efficiency is a function o f boiling time. There is an indication that the relationship levels off between 30 to 45 minutes o f boiling. Considering this trend in conjunction with the need for rapid assay turnaround, it was decided to use a 30 minute boiling step to effect lysis for certain subsequent
experiments (the 'no lysis' method was used unless otherwise stated). Notably, in this experiment the ratio o f the recovery o f pQR701 after 30 minutes o f boiling compared to the ‘no lysis’ control method is 20:1 (in other experiments the ratio has varied from 3:1 to 20:1). This suggests a substantial improvement in the sensitivity o f the PCR.
o s- Cl 'o
1
2 5 0 0 2 0 0 0- 2 _____ § 1 5 0 0 - o 1 000- cts 5 0 0 -œ ntrol 10 min 2 0 min 3 0 min 4 5 min
boiling time
FIGURE 4.2 Effect o f v a r\in g boiling tim e on lysis efficiency. F igure show s recoveries o f plasm id pQR701 relative to control \ aliie (assigned 100%). V alues show n are averages (n = 2) ± SEM. B oiling was carried out using 2.5 x 10^ E. coli JM 107 pQR701 cells in
100 p L thiosulphate ringers solution, o f w hich 2 x 10 p L w as assayed by PCR.
It should also be noted that the 30 minute boiling step has an insignificant effect on the concentration o f extracellular (filtered) pQRTOl. This implies that heat does not cause
excessive DNA degradation, a point noted by Zwadyk el al (1994) who found that the
effect o f heating on M ycohacleriuni genomic DNA is to produce strands which are
suitable for PCR amplification. Additionally, this result suggests that the proportion o f pQRTOl that is extracellular in a culture would be overestimated if the ‘no lysis’ method is used (Section 5.3.2.1).
It is also w orth noting at this point that the PCR developed for a chromosomal E. coli
gene, g u s lf was intended to be used to determine absolute lysis efficiency effected by a
variety o f treatments. It was envisaged that since it is possible to determine the number o f chromosomes per cell under various grow th conditions (Bremer and Dennis, 198T) and the number o f cells in a sample, then it would be possible to calculate the total
Chapter 4. Adaptation o f the QPCR method for air sampling
number of copies of the gusR gene that should be made available to PCR after a theoretical 100 % efficient lysis procedure. Absolute rather than relative lysis efficiencies could then be determined. Unfortunately, it has not been possible to pursue these experiments in the time allowed.